Hematopoietic stem and progenitor cells derived from hemogenic endothelial cells by episomal plasmid gene transfer

ABSTRACT

Embodiments herein relate to in vitro production methods of hematopoietic stem cell (HSC) and hematopoietic stem and progenitor cell (HSPC) that have long-term multilineage hematopoiesis potentials upon in vivo engraftment. The HSC and HSPCs are derived from pluripotent stem cells-derived hemogenic endothelia cells (HE) by non-integrative episomal vectors-based gene transfer.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims benefit under 35 U.S.C. § 119(e) of the U.S.Provisional Application No. 62/519,412 filed Jun. 14, 2017, the contentsof which are incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant No.:R37AI039394, R24DK092760, and UO1-HL100001 awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

FIELD OF THE DISCLOSURE

This disclosure relates to in vitro production methods of hematopoieticstem cell (HSC) and hematopoietic stem and progenitor cell (HSPC)starting from hemogenic endothelia cells (HE) that were induced frompluripotent stem cells, including induced pluripotent stem cells (iPSC),and also relates to long-term multilineage hematopoiesis with theengraftment of these HSCs and HSCPs.

BACKGROUND

There is a lack of supply of functional blood cells for in vivo cellularreplacement therapy, and for in vitro studies of disease modeling, drugscreening, and hematological diseases. Bone marrow transplantation is byfar the most established cellular replacement therapy for a variety ofhematological disorders. The functional unit of a bone marrow transplantis the hematopoietic stem cell (HSC), which resides at the apex of acomplex cellular hierarchy and replenishes blood development throughoutlife. However, the scarcity of HLA-matched HSCs or patient-specific HSCsseverely limits the ability to carry out transplantation, diseasemodeling, drug screening, and in vitro studies of hematologicaldiseases. Often, there is not a large enough cell populationtransplanted to ensure sufficient engraftment and reconstitution invivo.

As such, many studies have been developed to generate HSCs fromalternative sources. For example, reprogramming of somatic cells toinduced pluripotent stem cells (iPSC) has provided access to a widearray of patient-specific pluripotent cells, a promising source fordisease modeling, drug screens and cellular therapies. Pluripotent cellsare induced in human and mouse somatic cells by the forced expression ofthe reprogramming factors: OCT4 (Oct4) and SOX2 (Sox2) with either thecombinations of KLF4 (Klf4) and c-MYC (c-Myc) or NANOG (Nanog) and LIN28(Lin28). Mouse iPS cell lines derived from bone marrow hematopoieticprogenitor cells has been reported. Derivation of human iPS cells frompostnatal human blood cells, from granulocyte colony-stimulating factor(G-CSF) mobilized peripheral blood (PB) CD34+ cells, and from human cordblood (CB) and adult bone marrow (BM) CD34+ cells without anypre-treatment such as G-CSF mobilization has been also reported. TheiPSCs have been shown to differentiate into various cells belonging tothe three germ layers, as demonstrated by the analysis of teratomasgenerated from human and mouse iPS cells. In addition, the pluripotencyof iPS cells is confirmed by the contribution of iPS cell-derived cellsto various organs of the chimeric mice developed from iPScell-introduced blastocysts.

Another approach to generate HSCs from pluripotent stem cells (PSC) isto specify HSCs from its ontogenetic precursors. It is now widelyaccepted that HSCs originate from hemogenic endothelium (HE) in theaorta-gonad-mesonephros (AGM) and arterial endothelium in otheranatomical sites. Recent work on the directed differentiation of HE fromhuman PSC have provided valuable insights into some of the signalingpathways that control the emergence of primitive or definitivepopulations; however, the endothelial-to-hematopoietic transitionremains incompletely understood in human hematopoietic development,making rational intervention challenging. For example, there are reportsof induced definitive HE differentiated from human embryonic bodies (EB)that were derived from iPS cells. However, these HE from PSCs do notengraft in vivo. In contrast, other have shown that real hemogenic cellsfrom human fetal tissues can engraft in mice, indicating missing signalsto confer HSC fate on PSCs.

Therefore, there are still barriers to the generation of HSC from thesealternative sources. In addition to the cell quantity and cell sourceproblems, there is still a hurdle in producing hematopoietic stem andprogenitor cells derived from human pluripotent stem cells (hPSCs) orthe differentiated cells therefrom that would engraft in vivo. Mostly,the primitive HSC and HSPC produced from these alternative sources donot sustain blood production in vivo.

SUMMARY OF THE DISCLOSURE

It is difficult to harvest de novo enough hematopoietic stem cells(HSCs) and hematopoietic stem and progenitor cells (HSPCs) from animalsand humans. It is also difficult to ex vivo culture expand enough ofthese cells for any meaningful therapeutic purposes. Sometimes, the exvivo culture-expanded cells do not differentiate into all thehematopoietic lineage potentials.

Additionally, it is difficult to differentiate HSCs and HSPCs frompluripotent stem cells (PSCs) where the HSCs and HSPCs exhibit all thehematopoietic lineage potentials. One of the most common problems withHSCs and HSPCs derived from PSCs is that the HSCs and HSPCs do notengraft well and in sufficient number in the host after transplantationto sustain blood production in vivo. One of the problems to solve inachieving in vivo long-term multilineage hematopoiesis with theengraftment of these HSCs and HSCPs from the PSCs.

The inventors have found a process to make PSCs-derived HSCs and HSPCsthat would differentiate into all the hematopoietic lineage potentialsand would also engraft well in the host after transplantation so thatthere is sufficient engrafted cells to sustain blood production in vivo.This discovery provides a method for producing functionally relevantHSCs and HSPCs in sufficient quantities for both meaningful experimentaland therapeutic purposes. For example, in vitro experiments, thesePSCs-derived HSCs and HSPCs can be differentiated to the desiredhematopoietic lineage, e.g., erythroid cells, lymphoid cells, andmyeloid cells, for further studies, e.g., drug studies. For example, inin vivo studies, these PSCs-derived HSCs and HSPCs would engraft in ahost, and differentiate into a variety of hematopoietic progeny cells,and reconstitute and populate the circulatory and immune system of thehost.

Embodiments of the present disclosure are based, in part, to thediscovery of a few key transcription factors that would bring about thedifferentiation of HSCs and HSPCs that are derived from pluripotent stemcells derived hemogenic endothelia cells (HE). First, the inventorsshowed that embryonic bodies (EB) are made from pluripotent stem cells,e.g., including induced pluripotent cells. Second, the HE are harvestedfrom the EB, and cultured to induce endothelial-to-hematopoietictransition (EHT) in vitro. Then, the HE cells are transfected withcoding gene sequences of at least the following transcription factors:ERG, HOXA9, HOXA5, LCOR and RUNX1, for the expression of the respectivetranscription factors, thereby to promote differentiation of the HE intoHSCs and HSPCs that exhibit all the hematopoietic lineage potentials.These multilineage HSCs and HSPCs engraft well in recipient host afterimplantation. Additionally, it is shown herein that the transfection ofthe transcription factors described herein (e.g., ERG, HOXA9, HOXA5,LCOR and RUNX1) via a non-integrative vector (e.g., an epsiomal vector)increasing the yield and efficacy of engratftment as compared to anintegrative vector (e.g., a lentivirus).

Accordingly, in one aspect, provided herein is a method for makinghematopoietic stem cells (HSCs) and hematopoietic stem and progenitorcells (HSPCs) comprising in vitro transfecting hemogenic endotheliacells (HE) with an exogenous gene coding copy of each of the followingtranscription factors: ERG, HOXA9, HOXA5, LCOR and RUNX1 comprised in anon-integrative vector, wherein the transcription factors are expressedin the transfected cells to produce a population of multilineage HSCsand HSPCs that engrafts in recipient host after implantation. Additionaltranscription factors, HOXA10 and SPI1, are optionally included.

In another aspect, this disclosure provides is a method of makinghematopoietic stem cells (HSCs) and hematopoietic stem and progenitorcells (HSPCs) comprising (a) generating embryonic bodies (EB) frompluripotent stem cells; (b) isolating hemogenic endothelia cells (HE)from the resultant population of EB; (c) inducingendothelial-to-hematopoietic transition (EHT) in culture in the isolatedHE in order to obtain hematopoietic stem cells, and (d) in vitrotransfecting the induced HE with an exogenous gene coding copy of eachof the following transcription factors ERG, HOXA9, HOXA5, LCOR and RUNX1comprised in a non-integrative vector. Additional transcription factors,HOXA10 and SPI1, are optionally included.

In another aspect, this disclosure provides is an engineered cellderived from a population of HE that is produced by a method comprising(a) generating embryonic bodies (EB) from pluripotent stem cells; (b)isolating hemogenic endothelia cells (HE) from the resultant populationof EB; (c) inducing endothelial-to-hematopoietic transition (EHT) inculture in the isolated HE in order to obtain hematopoietic stem cells,and (d) in vitro transfecting the population of HE with an exogenousgene coding copy of each of the following transcription factors ERG,HOXA9, HOXA5, LCOR and RUNX1 comprised in a non-integrative vector.Additional transcription factors, HOXA10 and SPI1, are optionallyincluded.

In another aspect, this disclosure provides is an engineered cellderived from a population of HE that is produced by a method comprisingin vitro transfecting the population of HE with an exogenous gene codingcopy of each of the following transcription factors ERG, HOXA9, HOXA5,LCOR and RUNX1. Additional transcription factors, HOXA10 and SPI1, areoptionally included.

In another aspect, this disclosure provides is an engineered cellcomprises an exogenous copy of each of the following transcriptionfactors ERG, HOXA9, HOXA5, LCOR and RUNX1. Additional transcriptionfactors, HOXA10 and SPI1, are optionally included.

In another aspect, this disclosure provides is a composition comprisinga population of engineered cells derived from a population of HE andproduced by a method comprising (a) generating embryonic bodies (EB)from pluripotent stem cells; (b) isolating hemogenic endothelia cells(HE) from the resultant population of EB; (c) inducingendothelial-to-hematopoietic transition (EHT) in culture in the isolatedHE in order to obtain hematopoietic stem cells, and (d) in vitrotransfecting the population of HE with an exogenous gene coding copy ofeach of the following transcription factors ERG, HOXA9, HOXA5, LCOR andRUNX1 comprised in a non-integrative vector. Additional transcriptionfactors, HOXA10 and SPI1, are optionally included. In some embodiments,this composition is useful for cellular replacement therapy in asubject. In other embodiments, this composition is useful for researchand laboratory uses. For examples, in drug screening and testing.

In another aspect, this disclosure provides is a composition comprisinga population of engineered cells derived from a population of HE andproduced by a method comprising in vitro transfecting the population ofHE with an exogenous gene coding copy of each of the followingtranscription factors ERG, HOXA9, HOXA5, LCOR and RUNX1. Additionaltranscription factors, HOXA10 and SPI1, are optionally included. In someembodiments, this composition is useful for cellular replacement therapyin a subject. In other embodiments, this composition is useful forresearch and laboratory uses.

In another aspect, this disclosure provides is a composition comprisinga population of engineered cells wherein the cells comprise an exogenousgene coding copy of each of the following transcription factors ERG,HOXA9, HOXA5, LCOR and RUNX1. Additional transcription factors, HOXA10and SPI1, are optionally included. Additionally, the engineered cellsfurther comprises reprogramming factors OCT4, SOX2, KLF4 and optionallyc-MYC or NANOG and LIN28.

In another aspect, this disclosure provides is a pharmaceuticalcomposition comprising a population of engineered cells derived from apopulation of HE and a pharmaceutically acceptable carrier, wherein theengineered cells are produced by a method comprising (a) generatingembryonic bodies (EB) from pluripotent stem cells; (b) isolatinghemogenic endothelia cells (HE) from the resultant population of EB; (c)inducing endothelial-to-hematopoietic transition (EHT) in culture in theisolated HE in order to obtain hematopoietic stem cells, and (d) invitro transfecting the population of HE with an exogenous gene codingcopy of each of the following transcription factors ERG, HOXA9, HOXA5,LCOR and RUNX1 comprised in a non-integrative vector. Additionaltranscription factors, HOXA10 and SPI1, are optionally included. In someembodiments, this pharmaceutical composition is useful for cellularreplacement therapy in a subject.

In another aspect, this disclosure provides is a pharmaceuticalcomposition comprising a population of engineered cells derived from apopulation of HE and a pharmaceutically acceptable carrier, wherein theengineered cells are produced by a method comprising in vitrotransfecting the population of HE with an exogenous gene coding copy ofeach of the following transcription factors ERG, HOXA9, HOXA5, LCOR andRUNX1. Additional transcription factors, HOXA10 and SPI1, are optionallyincluded. In some embodiments, this pharmaceutical composition is usefulfor cellular replacement therapy in a subject.

In another aspect, this disclosure provides is a pharmaceuticalcomposition comprising a population of engineered cells and apharmaceutically acceptable carrier, wherein the engineered cellscomprise an exogenous gene coding copy of each of the followingtranscription factors ERG, HOXA9, HOXA5, LCOR and RUNX1. Additionaltranscription factors, HOXA10 and SPI1, are optionally included.

In another aspect, this disclosure provides is a method of cellularreplacement therapy in a subject in need thereof, the method comprisingadministering a population of engineered cells to a recipient subject,the population of engineered cells are produced by a method comprising(a) generating embryonic bodies (EB) from pluripotent stem cells; (b)isolating hemogenic endothelia cells (HE) from the resultant populationof EB; (c) inducing endothelial-to-hematopoietic transition (EHT) inculture in the isolated HE in order to obtain hematopoietic stem cells,and (d) in vitro transfecting the population of HE with an exogenousgene coding copy of each of the following transcription factors ERG,HOXA9, HOXA5, LCOR and RUNX1 comprised in a non-integrative vector.Additional transcription factors, HOXA10 and SPI1, are optionallyincluded.

In another aspect, this disclosure provides is a method of cellularreplacement therapy in a subject in need thereof, the method comprisingadministering a population of engineered cells to a recipient subject,the population of engineered cells are produced by a method comprisingin vitro transfecting the population of HE with an exogenous gene codingcopy of each of the following transcription factors ERG, HOXA9, HOXA5,LCOR and RUNX1. Additional transcription factors, HOXA10 and SPI1, areoptionally included.

In another aspect, this disclosure provides is a method of cellularreplacement therapy in a subject in need thereof, the method comprisingadministering a population of engineered cells to a recipient subject,the population of engineered cells comprise an exogenous gene codingcopy of each of the following transcription factors ERG, HOXA9, HOXA5,LCOR and RUNX1. Additional transcription factors, HOXA10 and SPI1, areoptionally included.

In another aspect, this disclosure provides is an engineered cellderived from a population of HE and produced by a method describedherein.

In another aspect, this disclosure provides is a composition comprisinga population of engineered cells described herein.

In another aspect, this disclosure provides is a pharmaceuticalcomposition comprising a population of engineered cells described hereinand a pharmaceutically acceptable carrier.

In another aspect, this disclosure provides is a pharmaceuticalcomposition described herein for use in cellular replacement therapy ina subject.

In another aspect, this disclosure provides is a method of cellularreplacement therapy in a subject in need thereof, the method comprisingadministering a population of engineered cells described, or acomposition described, or a pharmaceutical composition described to arecipient subject.

In another aspect, this disclosure provides is a use of an engineeredcell described herein, a composition comprising of engineered cellsdescribed herein, or a pharmaceutical composition comprising ofengineered cells described herein for the cellular replacement therapyin a subject in need thereof, or for the manufacture of medicament forcellular replacement therapy in a subject in need thereof.

In one embodiment of any one aspect described, the method described isan in vitro method.

In one embodiment of any one aspect described, the EB are generated orinduced from PSC by culturing or exposing the PSC to mophogens for about8 days.

In one embodiment of any one aspect described, the mophogens forgenerating EBs from PSC are selected from the group consisting ofholo-transferrin, mono-thioglycerol (MTG), ascorbic acid, bonemorphogenetic protein (BMP)-4, basic fibroblast growth factor (bFGF),SB431542, CHIR99021, vascular endothelial growth factor (VEGF),interleukin (IL)-6, insulin-like growth factor (IGF)-1, interleukin(IL)-11, stem cell factor (SCF), erythropoietin (EPO), thrombopoietin(TPO), interleukin (IL)-3, and Fms related tyrosine kinease 3 ligand(Flt-3L). The combination of all these factors required to producedefinitive HE.

In one embodiment of any one aspect described, the method describedherein further comprises selecting EBs that are formed from the PSCafter having been exposed or contacted with the described morphogens.

In one embodiment of any one aspect described, the selected EBs are lessthan 800 microns in size and are selected.

In one embodiment of any one aspect described, the EB cells within theselected EBs are compactly adhered to each other and requires trypsindigestion in order to dissociate the cells to individual cells.

In one embodiment of any one aspect described, the EB cells of theselected EBs are dissociated prior to the isolation of HE therefrom.

In one embodiment of any one aspect described, the population of PSCused for generating EBs is induced pluripotent stem cells (iPS cells) orembryonic stem cells (ESC).

In one embodiment of any one aspect described, the iPS cells areproduced by introducing only reprogramming factors OCT4, SOX2, and KLF4,and optionally c-MYC into mature cells. In one embodiment of any oneaspect described, the iPS cells are produced by introducing onlyreprogramming factors OCT4, SOX2, and KLF4, and optionally NANOG andLIN28 into mature cells. The introduction is via any method known in thearts, e.g., viral vectors (AAV, lentiviral, retroviral vectors), orother non-integrative episomal vectors (oriP/EBNA-1 [Epstein Barrnuclear antigen-1], the non-viral episomal vector pEPI-1) that are knownin the art.

In one embodiment of any one aspect described, the mature cells forproducing iPS cells are selected from the group consisting of Blymphocytes (B-cells), T lymphocytes, (T-cells), fibroblasts, andkeratinocytes. Any matured, differentiated cells in the body of amulticellular organism can be used to produce iPCs.

In one embodiment of any one aspect described, the induced pluripotentstem cells are produced by introducing the reprogramming factors once,or two or more times into the mature cells.

In one embodiment of any one aspect described, the disclosedtranscription factors are expressed in the transfected cells, that is,the respective transcription factors: ERG, HOXA9, HOXA5, LCOR, RUNX1,HOXA10, or SPI1, is expressed in the transfected cells. In oneembodiment, the respective transcription factors: ERG, HOXA9, HOXA5,LCOR, RUNX1, HOXA10, or SPI1, is expressed in the transfected cells viaa non-integrative vector.

In one embodiment, the non-integrative vector is an episomal vector.

In one embodiment, at least 2, at least 3, at least 4, or at least 5transcription factors are transfected.

In another embodiment of any one aspect described, the disclosedtranscription factors are expressed in the engineered cells of thisdisclosure.

In one embodiment of any one aspect described, the expression of thedisclosed transcription factors in the transfected or engineered cellsproduces a population of multi-lineage HSCs and HSPCs.

In one embodiment of any one aspect described, the population ofmulti-lineage HSCs and HSPCs, produced by the expression of thedisclosed transcription factors in the transfected or engineered cells,engrafts in vivo in the recipient subject and produces blood cells invivo.

In one embodiment of any one aspect described, the population ofmultilineage HSCs and HSPCs, produced by the expression of the disclosedtranscription factors in the transfected or engineered cells,reconstitutes the hematopoietic system in vivo in the recipient subject.

In one embodiment of any one aspect described, the population ofmulti-lineage HSCs and HSPCs differentiate to myeloid cells in vivoafter implantation in a host recipient subject. The myeloid cellsproduce MPO upon PMA or cytokine stimulation in vivo.

In one embodiment of any one aspect described, the population ofmulti-lineage HSCs and HSPCs differentiate to functional T- and B-cellsin vivo after implantation in a host recipient subject. The functionalT- and B-cells produce IgM and IgG. The functional T- and B-cells alsoundergo immunoglobulin class switching in response to ovalbuminstimulation. The functional T- and B-cells also produces INF-γ.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein express at least one of the following transcriptionfactors: ERG, HOXA9, HOXA5, LCOR, RUNX1, HOXA10, or SPI1, from anexogenous gene encoding the transcription factors in the cells.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are multilineage HSCs and HSPCs that are CD34+.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are multilineage HSCs and HSPCs that are CD34+ andCD45+.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are multilineage HSCs and HSPCs that are CD34+CD45+ andCD38−.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are CD34+.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are are CD34+ and CD45+.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are CD34+CD45+ and CD38−.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are multilineage HSCs and HSPCs that engraft in vivo ina host recipient subject and produce blood cells in vivo.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are multilineage HSCs and HSPCs that reconstitutes thehematopoietic system in vivo when transplanted into a host recipientsubject.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are multilineage HSCs and HSPCs that differentiate tomyeloid cells in vivo, and the myeloid cells produce MPO upon PMA orcytokine stimulation in vivo.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are multilineage HSCs and HSPCs that differentiate tofunctional T- and B-cells in vivo, the functional T- and B-cells produceIgM and IgG. The functional T- and B-cells also undergo immunoglobulinclass switching in response to ovalbumin stimulation. The functional T-and B-cells also produces INF-γ.

In one embodiment of any one aspect described, the HE are definitive HE.

In one embodiment of any one aspect described, the HE are FLK1+, CD34+,CD43−, and CD235A−. These biomarkers are those on HE before theendothelial-to-hematopoietic transition (EHT).

Definitive HE is a population that is defined by combination of surfaceantigen markers. CD34+FLK1+CD235A−CD43− before EHT.

In one embodiment of any one aspect described, the HE are isolatedimmediately from selected EBs and dissociated EB cells.

In one embodiment of any one aspect described, the HSCs are CD34+.

In one embodiment of any one aspect described, the HSCs are CD34+ andCD45+.

In one embodiment of any one aspect described, the HSPCs are CD34+.

In one embodiment of any one aspect described, the HSPCs are CD34+ andCD45+.

In one embodiment of any one aspect described, the EHT occurs byculturing the isolated HE in thrombopoietin (TPO), interleukin (IL)-3,stem cell factor (SCF), IL-6, IL-11, insulin-like growth factor (IGF)-1,erythropoietin (EPO), vascular endothelial growth factor (VEGF), basicfibroblast growth factor (bFGF), bone morphogenetic protein (BMP)4, Finsrelated tyrosine kinase 3 ligand (Flt-3L), sonic hedgehog (SHH),angiotensin II, and chemical AGTR1 (angiotensin II receptor type I)blocker losartan potassium.

In one embodiment of any one aspect described, the multilineage HSCsproduced by the methods described in this disclosure areCD34+CD38−CD45+.

In one embodiment of any one aspect described, the multilineage HSPCsproduced by the methods described in this disclosure are CD34+CD45+.

In one embodiment of any one aspect described, the engineered cell ofthis disclosure comprises an exogenous copy of each of the followingtranscription factors ERG, HOXA9, HOXA5, LCOR and RUNX1.

In one embodiment of any one aspect described, the engineered cell ofthis disclosure further comprises an exogenous copy of each of thefollowing reprogramming factors OCT4, SOX2, KLF4 and optionally c-MYC.Alternate reprogramming factors in lieu of c-MYC are NANOG and LIN28.

In one embodiment of any one aspect described, the composition ofengineered cells of this disclosure further comprises a pharmaceuticallyacceptable carrier.

In one embodiment of any one aspect described, the subject is a patientwho has undergone chemotherapy or irradiation or both chemotherapy andirradiation, and manifest deficiencies in immune or blood function orlymphocyte reconstitution or both deficiencies in immune function andlymphocyte reconstitution.

In one embodiment of any one aspect described, the subject prior toimplantation, the immune cells are treated ex vivo with prostaglandin E2and/or antioxidant N-acetyl-L-cysteine (NAC) to promote subsequentengraftment in a recipient subject.

In one embodiment of any one aspect described, the engineered cells ofthis disclosure are autologous to the recipient subject or at least HLAtype matched with the recipient subject.

Definitions

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

The term “consisting of” refers to methods, and respective componentsthereof as described herein, which are exclusive of any element notrecited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the disclosure.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike. A pharmaceutically acceptable carrier will not promote the raisingof an immune response to an agent with which it is admixed, unless sodesired. The preparation of a pharmacological composition that containsactive ingredients dissolved or dispersed therein is well understood inthe art and need not be limited based on formulation. Typically suchcompositions are prepared as injectable either as liquid solutions orsuspensions, however, solid forms suitable for solution, or suspensions,in liquid prior to use can also be prepared. The preparation can also beemulsified or presented as a liposome composition. The active ingredientcan be mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient and in amounts suitable for use inthe therapeutic methods described herein. Suitable excipients include,for example, water, saline, dextrose, glycerol, ethanol or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents and the like which enhance theeffectiveness of the active ingredient. The therapeutic composition ofthe embodiments of the present disclosure can include pharmaceuticallyacceptable salts of the components therein. Pharmaceutically acceptablesalts include the acid addition salts (formed with the free amino groupsof the polypeptide) that are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, tartaric, mandelic and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like. Physiologically tolerablecarriers are well known in the art. Exemplary liquid carriers aresterile aqueous solutions that contain no materials in addition to theactive ingredients and water, or contain a buffer such as sodiumphosphate at physiological pH value, physiological saline or both, suchas phosphate-buffered saline. Still further, aqueous carriers cancontain more than one buffer salt, as well as salts such as sodium andpotassium chlorides, dextrose, polyethylene glycol and other solutes.Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions. The amount of an active agent used in the methods describedherein that will be effective in the treatment of a particular disorderor condition will depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques. Suitablepharmaceutical carriers are described in Remington's PharmaceuticalSciences, A. Osol, a standard reference text in this field of art. Forexample, a parenteral composition suitable for administration byinjection is prepared by dissolving 1.5% by weight of active ingredientin 0.9% sodium chloride solution.

In one embodiment, the “pharmaceutically acceptable” carrier does notinclude in vitro cell culture media.

In one embodiment, the term “pharmaceutically acceptable” means approvedby a regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, and more particularly in humans. Specifically, it refers tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the therapeutic is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations, andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in Remington's Pharmaceutical Sciences, 18th Ed., Gennaro,ed. (Mack Publishing Co., 1990). The formulation should suit the mode ofadministration.

A “subject,” as used herein, includes any animal that exhibits a symptomof a monogenic disease, disorder, or condition that can be treated withthe cell-based therapeutics, and methods disclosed elsewhere herein. Inone embodiment, a subject includes any animal that exhibits symptoms ofa disease, disorder, or condition of the hematopoietic system, e.g., ahemoglobinopathy, that can be treated with the cell-based therapeutics,and methods contemplated herein. Suitable subjects (e.g., patients)include laboratory animals (such as mouse, rat, rabbit, or guinea pig),farm animals, and domestic animals or pets (such as a cat or dog).Non-human primates and, preferably, human patients, are included.Typical subjects include animals that exhibit aberrant amounts (lower orhigher amounts than a “normal” or “healthy” subject) of one or morephysiological activities that can be modulated by gene therapy.

In one embodiment, as used herein “treatment” or “treating,” includesany beneficial or desirable effect on the symptoms or pathology of adisease or pathological condition, and may include even minimalreductions in one or more measurable markers of the disease or conditionbeing treated. In another embodiment, treatment can involve optionallyeither the reduction or amelioration of symptoms of the disease orcondition, or the delaying of the progression of the disease orcondition. “Treatment” does not necessarily indicate completeeradication or cure of the disease or condition, or associated symptomsthereof.

As used herein, the term “amount” refers to “an amount effective” or “aneffective amount” of transduced therapeutic cells to achieve abeneficial or desired prophylactic or therapeutic result, includingclinical results.

A “prophylactically effective amount” refers to an amount of transducedtherapeutic cells effective to achieve the desired prophylactic result.Typically, but not necessarily, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount is less than the therapeuticallyeffective amount.

A “therapeutically effective amount” of transduced therapeutic cells mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the stem and progenitorcells to elicit a desired response in the individual. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the virus or transduced therapeutic cells are outweighed by thetherapeutically beneficial effects. The term “therapeutically effectiveamount” includes an amount that is effective to “treat” a subject (e.g.,a patient).

As used herein, the terms “administering,” refers to the placement of acomposition or engineered cells of this disclosure into a subject by amethod or route which results in at least a desired effects, forexample, increase number of immune cells or blood cells or platelets.The composition or engineered cells of this disclosure can beadministered by any appropriate route which results in an effectivetreatment in the subject.

As used herein, in one embodiment, the term “hematopoietic stem cell” or“HSC” refers to a stem cell that give rise to all the blood cell typesof the three hematopoietic lineages, erythroid, lymphoid, and myeloid.These cell types include the myeloid lineages (monocytes andmacrophages, neutrophils, basophils, eosinophils, erythrocytes,megakaryocytes/platelets, dendritic cells), and the lymphoid lineages(T-cells, B-cells, NK-cells). In one embodiment, the term “hematopoieticstem cell” or “HSC” refers to a stem cell that have the following cellsurface markers: CD34+, CD59+, Thy1/CD90+, CD38lo/−, and C-kit/CD117+.In one embodiment, the term “hematopoietic stem cell” or “HSC” refers toa stem cell that is at least CD34+. In one embodiment, the term“hematopoietic stem cell” or “HSC” refers to a stem cell that is atleast CD34+ and C-kit/CD117+. In another embodiment, the term HSC refersto a stem cell that is at least CD34+. In another embodiment, the termHSC refers to a stem cell that is at least CD34+/CD45+. In anotherembodiment, the term HSC refers to a stem cell that is at leastCD34+/CD45+/CD38−.

As used herein, the terms “iPS cell” and “induced pluripotent stem cell”are used interchangeably and refers to a pluripotent cell artificiallyderived by the transfection of the following reprogramming factors OCT4,SOX2, KLF4 and optionally c-MYC or optionally NANOG and LIN28, into aundifferentiated cell from a differentiated cell.

As used herein, the term “lineage” when used in the context of stem andprogenitor cell differentiation and development refers to the celldifferentiation and development pathway which the cell can take tobecoming a fully differentiated cell. For example, a HSC has threehematopoietic lineages, erythroid, lymphoid, and myeloid; the HSC hasthe potential, ie., the ability, to differentiate and develop into thoseterminally differentiated cell types known for all these three lineages.When the term “multilineage” used, it means the cell is able in thefuture differentiate and develop into those terminally differentiatedcell types known for more than one lineage. For example, the HSC hasmultilineage potential. When the term “limited lineage” used, it meansthe cell can differentiate and develop into those terminallydifferentiated cell types known for one lineage. For example, a commonmyeloid progenitor cell or a megakaryocyte-erythroid progenitor has alimited lineage because the cell can only differentiate and develop intothose terminally differentiated cell types of the myeloid lineage andnot that of the lymphoid lineage. Terminally differentiated cells of themyeloid lineage include erythrocytes, monocytes, macrophages,megakaryocytes, myeloblasts, dendritic cells, and granulocytes(basophils, neutrophils, eosinophils, and mast cells); and terminallydifferentiated cells of the lymphoid lineage include T lymphocytes/Tcells, B lymphocytes/B cells, and natural killer cells.

As used herein, the term “a progenitor cell” refers to an immature orundifferentiated cell that has the potential later on to mature(differentiate) into a specific cell type, for example, a blood cell, askin cell, a bone cell, or a hair cells. Progenitor cells have acellular phenotype that is more primitive (e.g., is at an earlier stepalong a developmental pathway or progression than is a fullydifferentiated cell) relative to a cell which it can give rise to bydifferentiation. Often, progenitor cells also have significant or veryhigh proliferative potential. Progenitor cells can give rise to multipledistinct differentiated cell types or to a single differentiated celltype, depending on the developmental pathway and on the environment inwhich the cells develop and differentiate. A progenitor cell also canproliferate to make more progenitor cells that are similarly immature orundifferentiated.

As used herein, the term “multilineage hematopoietic progenitor cells”or “hematopoietic stem and progenitor cells (HSPC)” refer tohematopoietic cells (cell that form the blood) that have the ability orpotential to generate, or differentiate into, multiple types ofhematopoietic lineage cells.

As used herein, the term “long-term” when used in the context of“multilineage hematopoiesis” refers to in vivo implanted HSCs and/orHSPCs being capable of producing the three hematopoietic lineage cells,erythroid, lymphoid, and myeloid, for up to at least 12 weeks posttransplantation.

As used herein, the term “multilineage hematopoiesis” in the context ofHSCs and/or HSPCs refers to these cells capable of producing at leastthe three hematopoietic lineage cells, erythroid, lymphoid, and myeloid.

The term “differentiated cell” is meant any primary cell that is not, inits native form, pluripotent as that term is defined herein. The term a“differentiated cell” also encompasses cells that are partiallydifferentiated, such as multipotent cells (e.g. adult somatic stemcells). In some embodiments, the term “differentiated cell” also refersto a cell of a more specialized cell type derived from a cell of a lessspecialized cell type (e.g., from an undifferentiated cell or areprogrammed cell) where the cell has undergone a cellulardifferentiation process.

In the context of cell ontogeny, the term “differentiate”, or“differentiating” is a relative term meaning a “differentiated cell” isa cell that has progressed further down the developmental pathway thanits precursor cell. Thus in some embodiments, a reprogrammed cell asthis term is defined herein, can differentiate to lineage-restrictedprecursor cells (such as a mesodermal stem cell or a endodermal stemcell), which in turn can differentiate into other types of precursorcells further down the pathway (such as an tissue specific precursor,for example, a cardiomyocyte precursor, or a pancreatic precursor), andthen to an end-stage differentiated cell, which plays a characteristicrole in a certain tissue type, and may or may not retain the capacity toproliferate further.

The term “multipotent” when used in reference to a “multipotent cell”refers to a cell that is able to differentiate into some but not all ofthe cells derived from all three germ layers. Thus, a multipotent cellis a partially differentiated cell. Multipotent cells are well known inthe art, and examples of muiltipotent cells include adult somatic stemcells, such as for example, hematopoietic stem cells and neural stemcells, hair follicle stem cells, liver stem cells etc. Multipotent meansa stem cell may form many types of cells in a given lineage, but notcells of other lineages. For example, a multipotent blood stem cell canform the many different types of blood cells (red, white, platelets,etc. . . . ), but it cannot form neurons; cardiovascular progenitor cell(MICP) differentiation into specific mature cardiac, pacemaker, smoothmuscle, and endothelial cell types; pancreas-derived multipotentprogenitor (PMP) colonies produce cell types of pancreatic lineage(cells that produces insulin, glucagon, amylase or somatostatin) andneural lineage (cells that are morphologically neuron-like,astrocytes-like or oligodendrocyte-like).

The term a “reprogramming gene”, as used herein, refers to a gene whoseexpression, contributes to the reprogramming of a differentiated cell,e.g. a somatic cell to an undifferentiated cell (e.g. a cell of apluripotent state or partially pluripotent state). A reprogramming genecan be, for example, genes encoding master transcription factors Sox2,Oct3/4, Klf4, Nanog, Lin-28, c-myc and the like. The term “reprogrammingfactor” refers to the protein encoded by the reprogramming gene.

The term “exogenous” refers to a substance present in a cell other thanits native source. The terms “exogenous” when used herein refers to anucleic acid (e.g. a nucleic acid encoding a reprogramming transcriptionfactor, e.g. Sox2, Oct3/4, Klf4, Nanog, Lin-28, c-myc and the like) or aprotein (e.g., a transcription factor polypeptide) that has beenintroduced by a process involving the hand of man into a biologicalsystem such as a cell or organism in which it is not normally found orin which it is found in lower amounts. A substance (e.g. a nucleic acidencoding a sox2 transcription factor, or a protein, e.g., a SOX2polypeptide) will be considered exogenous if it is introduced into acell or an ancestor of the cell that inherits the substance.

The term “isolated” as used herein signifies that the cells are placedinto conditions other than their natural environment. The term“isolated” does not preclude the later use of these cells thereafter incombinations or mixtures with other cells.

As used herein, the term “expanding” refers to increasing the number oflike cells through cell division (mitosis). The term “proliferating” and“expanding” are used interchangeably.

As used herein, a “cell-surface marker” refers to any molecule that isexpressed on the surface of a cell. Cell-surface expression usuallyrequires that a molecule possesses a transmembrane domain. Somemolecules that are normally not found on the cell-surface can beengineered by recombinant techniques to be expressed on the surface of acell. Many naturally occurring cell-surface markers are termed “CD” or“cluster of differentiation” molecules. Cell-surface markers oftenprovide antigenic determinants to which antibodies can bind to. Acell-surface marker of particular relevance to the methods describedherein is CD34. The useful hematopoietic progenitor cells according tothe present disclosure preferably express CD34 or in other words, theyare CD34 positive.

A cell can be designated “positive” or “negative” for any cell-surfacemarker, and both such designations are useful for the practice of themethods described herein. A cell is considered “positive” for acell-surface marker if it expresses the marker on its cell-surface inamounts sufficient to be detected using methods known to those of skillin the art, such as contacting a cell with an antibody that bindsspecifically to that marker, and subsequently performing flow cytometricanalysis of such a contacted cell to determine whether the antibody isbound the cell. It is to be understood that while a cell may expressmessenger RNA for a cell-surface marker, in order to be consideredpositive for the methods described herein, the cell must express it onits surface. Similarly, a cell is considered “negative” for acell-surface marker if it does not express the marker on itscell-surface in amounts sufficient to be detected using methods known tothose of skill in the art, such as contacting a cell with an antibodythat binds specifically to that marker and subsequently performing flowcytometric analysis of such a contacted cell to determine whether theantibody is bound the cell. In some embodiments, where agents specificfor cell-surface lineage markers used, the agents can all comprise thesame label or tag, such as fluorescent tag, and thus all cells positivefor that label or tag can be excluded or removed, to leave uncontactedhematopoietic stem or progenitor cells for use in the methods describedherein.

As used herein, “reprogramming factors” refers to factors used todedifferentiate a cell population. A number of such factors are known inthe art, for example, a set of transcription factors that have beenidentified to, e.g., promoting dedifferentitation. Exemplaryreprogramming factors include, but are not limited to Oct3, Sox1, Sox2,Sox3, Sox15, Klf1, Klf2, Klf4, Klf5, c-Myc, L-Myc, N-Myc, Nanog, Lin-28,SV40LT, Glis1, and p53 shRNA. In one embodiment, a reprogramming factoris an environmental condition, such as serum starvation.

The term “vector”, as used herein, refers to a nucleic acid constructdesigned for delivery to a host cell or for transfer between differenthost cells. As used herein, a vector can be viral or non-viral.

The term “vector” encompasses any genetic element that is capable ofreplication when associated with the proper control elements and thatcan transfer gene sequences to cells. A vector can include, but is notlimited to, a cloning vector, an expression vector, a plasmid, phage,transposon, cosmid, artificial chromosome, virus, virion, etc.

As used herein, the term “viral vector” refers to a nucleic acid vectorconstruct that includes at least one element of viral origin and has thecapacity to be packaged into a viral vector particle. The viral vectorcan contain a nucleic acid encoding a polypeptide as described herein inplace of non-essential viral genes. The vector and/or particle may beutilized for the purpose of transferring nucleic acids into cells eitherin vitro or in vivo. Numerous forms of viral vectors are known in theart.

As used herein, the term “expression vector” refers to a vector thatdirects expression of an RNA or polypeptide (e.g., a polypeptideencoding SIRT1) from nucleic acid sequences contained therein linked totranscriptional regulatory sequences on the vector. The sequencesexpressed will often, but not necessarily, be heterologous to the cell.An expression vector may comprise additional elements, for example, theexpression vector may have two replication systems, thus allowing it tobe maintained in two organisms, for example in human cells forexpression and in a prokaryotic host for cloning and amplification. Theterm “expression” refers to the cellular processes involved in producingRNA and proteins and as appropriate, secreting proteins, including whereapplicable, but not limited to, for example, transcription, transcriptprocessing, translation and protein folding, modification andprocessing.

A vector can be integrating or non-integrating. “Integrating vectors”have their delivered RNA/DNA permanently incorporated into the host cellchromosomes. “Non-integrating vectors” remain episomal which means thenucleic acid contained therein is never integrated into the host cellchromosomes. Examples of integrating vectors include retrovirualvectors, lentiviral vectors, hybrid adenoviral vectors, and herpessimplex viral vector.

One example of a non-integrative vector is a non-integrative viralvector. Non-integrative viral vectors eliminate the risks posed byintegrative retroviruses, as they do not incorporate their genome intothe host DNA. One example is the Epstein Barr oriP/Nuclear Antigen-1(“EBNA1”) vector, which is capable of limited self-replication and knownto function in mammalian cells. As containing two elements fromEpstein-Barr virus, oriP and EBNA1, binding of the EBNA1 protein to thevirus replicon region oriP maintains a relatively long-term episomalpresence of plasmids in mammalian cells. This particular feature of theoriP/EBNA1 vector makes it ideal for generation of integration-freeiPSCs. Another non-integrative viral vector is adenoviral vector and theadeno-associated viral (AAV) vector.

Another non-integrative viral vector is RNA Sendai viral vector, whichcan produce protein without entering the nucleus of an infected cell.The F-deficient Sendai virus vector remains in the cytoplasm of infectedcells for a few passages, but is diluted out quickly and completely lostafter several passages (e.g., 10 passages).

Another example of a non-integrative vector is a minicircle vector.Minicircle vectors are circularized vectors in which the plasmidbackbone has been released leaving only the eukaryotic promoter andcDNA(s) that are to be expressed.

The term “lentivirus” refers to a group (or genus) of retroviruses thatgive rise to slowly developing disease. Viruses included within thisgroup include HIV (human immunodeficiency virus; including HIV type 1,and HIV type 2), the etiologic agent of the human acquiredimmunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis(visna) or pneumonia (maedi) in sheep, the caprinearthritis-encephalitis virus, which causes immune deficiency, arthritis,and encephalopathy in goats; equine infectious anemia virus, whichcauses autoimmune hemolytic anemia, and encephalopathy in horses; felineimmunodeficiency virus (FIV), which causes immune deficiency in cats;bovine immune deficiency virus (BIV), which causes lymphadenopathy,lymphocytosis, and possibly central nervous system infection in cattle;and simian immunodeficiency virus (SIV), which cause immune deficiencyand encephalopathy in sub-human primates. Diseases caused by theseviruses are characterized by a long incubation period and protractedcourse. Usually, the viruses latently infect monocytes and macrophages,from which they spread to other cells. HIV, FIV, and SIV also readilyinfect T lymphocytes, i.e., T-cells.

The term “promoter/enhancer” refers to a segment of DNA which containssequences capable of providing both promoter and enhancer functions. Forexample, the long terminal repeats of retroviruses contain both promoterand enhancer functions. The enhancer/promoter may be “endogenous,”“exogenous,” or “heterologous.” An “endogenous” enhancer/promoter is onewhich is naturally linked with a given gene in the genome. An“exogenous” or “heterologous” enhancer/promoter is one which is placedin juxtaposition to a gene by means of genetic manipulation (i.e.,molecular biological techniques) such that transcription of that gene isdirected by the linked enhancer/promoter.

A “nucleic acid,” as described herein, can be RNA or DNA, and can besingle or double stranded, and can be selected, for example, from agroup including: nucleic acid encoding a protein of interest, forexample, transcription factors and reprogramming factors describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F collectively demonstrate the in vivo screening of TFsconfers functional hematopoiesis.

FIG. 1A. hPSC-derived HE was cultured for additional 3 days in EHTmedia, then infected with library of 26 TFs. Cells were injectedintrafemorally into sublethally irradiated (250 rads) NSG mice andtreated with doxycycline for 2 weeks to induce transgene expression.

FIG. 1B. Engraftment of human CD45+ cells was determined by flowcytometry of BM at 12 weeks. Library (N=6), 7 TFs (N=15), and 5 TFPoly(N=8). Defined 7 TFs and 5 TFPoly confer robust engraftment of HE. Thechimerism of human CD45+ population in BM was measured at 12 weeks. HEtransduced with either lentiviral library of HSC-specific TFs (Library);defined set of RUNX1, ERG, SPI1, LCOR, HOXA5, HOXA9, and HOXA10 (7 TFs);defined set of RUNX1, ERG, LCOR, HOXA5, and HOXA9 in polycistronicvectors (5 TFPoly).

FIG. 1C. Multilineage contribution of donor-derived cells in BM. After14 weeks, BM of NSG mice engrafted with TF library was analyzed formyeloid (M; CD33+), erythroid (E; GLY-A+), B-(CD19+), and T-cells (CD3+)within the human CD45+ population. Each bar represents individualrecipients engrafted. From left, recipient ID#1, #5 and #6 engraftedwith hiPSCs; recipient ID#2 left (L) femur and right (R) femur,recipient ID#3 left (L) femur and right (R) femur engrafted with hESCs;recipient ID#1 and #2 engrafted with CB-HSCs as a reference.

FIGS. 1D-1E. Primary transplantation of BM engrafted with 7 TF (FIG. 1D)and 5TFPoly (FIG. 1F) at 12 weeks. Human CD45+ BM of engrafted NSG wasanalyzed for HSCs (CD34+CD38−), nucleated erythroid (GLY-A+SYTO60+),enucleated erythroid (GLY-A+SYTO60−), neutrophils (PECAM+CD15+), B-cells(IgM+CD19+), B progenitor cells (IgM-CD19+), Plasmacytoid lymphocytes(IgM-CD19+CD38++) and T-cells (CD3+/CD4, CD8).

FIG. 1F. Lineage distribution of myeloid (CD33+), erythroid (GLY-A+),B-cells (CD19+) and T-cells (CD3+) was shown as a bar graph ofindividual recipients (N=5 for 7 TFs; N=4 for 5TFPoly).

FIGS. 2A-2G collectively demonstrate the detection of TFs that confermulti-lineage engraftment.

FIG. 2A. Transgene detection in engrafted cells of secondary recipientsof 7 TFs.

FIG. 2B. In vivo factor-minus-one (FMO) approach of defined 7 TFs toidentify any required or unnecessary factors. BM of NSG was analyzed at8 weeks for chimerism of human CD45+ population. The absence of RUNX1(0.33-fold, p=0.037), ERG (0.40-fold, p=0.056), LCOR (0.23-fold,p=0.020), HOXA5 (0.37-fold, p=0.056) or HOXA9 (0.26-fold, p=0.026)reduced chimerism. Lentiviral vector with GFP was used as negativecontrol. N=6 (4 for GFP). * p<0.05.

FIGS. 2C-2D. Secondary transplantation of BM engrafted with defined 7TFs. After 8 weeks from primary transplantation, 2,000 human CD34+ BMcells were transplanted to secondary recipients, and followed up to 8-14weeks. Compared with primary, secondary recipients had 0.34-fold fewerHSCs (p=0.12), 0.45-fold less nucleated erythroid (p=0.30), 3.3-foldmore enucleated erythroid (p=0.11), 1.6-fold more neutrophils (p=0.18),1.1-fold more mature B-cells (p=0.42), 0.65-fold more immature B-cells(p=0.10), and 0.56-fold less T cells (p=0.18). N=3 (primary), 2(secondary).

FIGS. 2E-2F. Phenotypic characterization of HSC-like cells in BMengrafted with 7 TF HSPCs. Human CD45+ population from BM of NSG miceengrafted with 7 TF HSPCs at 8 weeks was analyzed for human CD34 andCD38.

FIG. 2G. The population of HSCs (CD34+CD38−CD45+) was analyzed forcell-cycle state by Ki67 and DAPI. HSCs from NSG engrafted with CB-HSCsat the same time point were used as reference. N=3. The percentage ofHSCs in human CD45+ BM was 0.24-fold (p=0.058) (g); G0-phase was0.22-fold (p=0.0025); G1-phase was 2.3-fold (p=0.0052); S/G2/M was2.5-fold (p=0.013) vs CB equivalents (h). * p<0.01; ** p<0.05.

FIGS. 3A-3H collectively demonstrate the functional characterization ofterminally differentiated cells.

FIG. 3A. Globin switching in engrafted erythroid cells. Human GLY-A+cells were isolated from lysed BM (to exclude enucleated cells) of NSGengrafted with 7 TF HSPCs at 8 weeks and analyzed by qRT-PCR torelatively quantify HBE, HBG and HBB transcripts. Cytospin image ofisolated GLY-A+cells are shown. CB; GLY-A+erythroid cells fromCB-engrafted in NSG BM, 7 TF HSPCs; GLY-A+erythroid cells from 7 TFHSPC-engrafted in NSG BM, 5F; GLY-A+erythroid cells from hPSCstransduced with ERG, RORA, HOXA9, SOX4 and MYB17.

FIG. 3B. Enucleation of engrafted erythroid cells. BM of NSG miceengrafted with defined 7 TF HSPCs at 8 weeks time point was analyzed forhuman GLY-A and SYTO60. Cytospin images of RBCs from GLY-A+ populationsseparated by SYTO60 nuclear staining. Cells were isolated from unlysedBM of NSG engrafted with 7 TF HSPCs. Examples of nucleated andenucleated RBCs were shown. N=3.

FIG. 3C. Phenotyping of neutrophils. Human CD45+ population from BM ofNSG mice engrafted with defined 7 TF HSPCs at 8 weeks was analyzed forhuman PECAM and CD15. Myeloperoxidase activity of isolatedCD45+PECAM+CD15+neutrophils was measured with or without PMAstimulation. Neutrophils from NSG engrafted with CB-HSCs were used asreference. The basal level of MPO of HE was 0.40-fold less than CB(p=0.036). PMA stimulation increased MPO production 2.5-fold (p=0.010)(CB) and 3.0-fold (p=0.10) (7 TF). Stimulated MPO production of 7 TF was0.47-fold (0.049) vs CB. * p<0.05. Data are from 2 independentexperiments with 3 technical replicates each time.

FIGS. 3D-3E. Measuring production of Ig in serum. Serum was isolatedfrom NSG mice engrafted with CB-HSCs or defined 7 TF HSPCs at 8 weeks(IgM) (FIG. 3D) and 14 weeks (IgG) (FIG. 3E). Production of IgM and IgG(ng/mL serum) was measured by ELISA. Serum from mock transplant and NSGengrafted with CB-HSCs was used as reference. The production of bothhuman IgM and IgG was detected and boosted by OVA in 7 TF HSPCs. *p<0.05. Data are from 2 independent experiments with 3 technicalreplicates each time.

FIG. 3F. Human CD3+ cells were isolated from BM of NSG mice engraftedwith CB-HSCs and defined 7 TF HSPCs at 8 weeks and cultured with orwithout PMA/Ionomycin stimulation for 6 hours, when production of IFNγwas measured by ELISA. CD3+ T-cells from NSG engrafted with CB-HSCs wereused as reference. The basal level of IFNγ of HE was 0.53-fold (p=0.073)vs CB. PMA stimulation increased IFNγ production 4.4-fold (p=0.17) (CB)and 3.0-fold (p=0.16) (HE). Stimulated IFNγ production of HE was0.36-fold (0.039) vs CB. IFNγ production from CB-HSCs and HE themselveswere shown as reference. * p<0.05. Data are from 2 independentexperiments with 3 technical replicates each time.

FIG. 3G. Flow cytometric phenotyping of T-cells from engrafted 7 TFHSPCs. BM and thymus were collected at 8 weeks and analyzed for T cellmarkers (CD4, CD8, CD3, TCRαβ and TCRγδ). TCR phenotyping of the CD3+population was shown. One out of 3 recipients showed the presence ofTCRγδ. N=3.

FIG. 3H. TCR rearrangement of T-cells showed clonal diversity inhiPSC-derived cells. Human CD3+thymocytes of NSG mice engrafted withdefined 7 TF HSPCs at 8 weeks was analyzed to detect TCR rearrangementby Immuno-seq. CD3+Thymocytes from CB CD34+-engrafted NSG were used as areference.

FIG. 4. Scheme of in vivo screening of transcription factors (TFs) toconfer functional hematopoiesis in hPSC-derived hemogenicendothelium(HE). Human ESCs/iPSCswere differentiated to embryoidbodies (EBs) bycytokines for specification of hemogenicendothelium (HE). At day 8 timepoint, CD34+FLK1+CD43−CD235A-HE was isolated, and cultured inendothelial-to-hematopoietic transition (EHT) media for additional 3days. Library of HSC-specific TFs was induced in HE via lentivirus andinjected to sub-lethally irradiated immunodeficient NSG miceintrafemoraly. Engrafted hematopoietic cells were isolated and analyzedby genomic PCR to detect integrated TFs (positive hits).

FIG. 5. Venn diagram of TFs that conferred in vivo engraftment and invitro CFU on PSC-HE.

FIGS. 6A-6E show episomal-5TF-derived HSPCs show long-term multi-lineageengraftment in vivo. (FIG. 6A) Schematic illustration of the strategyfollowed for HSPC generation. (FIG. 6B) Percentage of human CD45⁺ cellsdetected by flow cytometry in bone marrow of injected leg of miceanalyzed at 10 (lenti-5TF n=12, epi-5TF n=12 and cord blood n=9 mice)and 16 weeks (lenti-5TF n=12, epi-5TF n=11 and cord blood n=9 mice) posttransplantation. (FIG. 6C) Percentage of human CD45⁺ cells detected byflow cytometry in injected and contralateral leg of engrafted mice at 10(lenti-5TF n=4, epi-5TF n=7 and cord blood n=9 mice) and 16 weeks(lenti-5TF n=4, epi-5TF n=9 and cord blood n=7 mice) posttransplantation. Line indicates 0.01% of human chimerism. L1-L8 weretransplanted with cells derived from hPSCs infected with lentiviralvectors (pINDUCER-21-L95 and pINDUCER-21-RE). E1-E16 were transplantedwith cells derived from hPSCs transfected with episomal vectors(pCXLE-L95, pCXLE-RE, pCXLE-EGFP and pCXWB-EBNA1). CB1-CB16 weretransplanted with human CD34⁺ umbilical cord blood cells. In grey isrepresented the engraftment in injected leg and in orange engraftment inthe contralateral leg. (FIG. 6D) Lineage distribution of myeloid (M;CD33+), B (B; CD19⁺) and T (T; CD3⁺) cells within the human CD45⁺population of injected and contralateral leg's bone marrow from primaryengrafted mice (>0.01% of human chimerism) analyzed by flow cytometry at10 and 16 weeks post injection. Injected leg is indicated as “I” andcontralateral leg is indicated as “C”. Mice with multi-lineageengraftment are indicated with an asterisk. (FIG. 6E) FACS plots showingrepresentative engraftment in bone marrow of a primary mousetransplanted with cells derived from hPSCs transfected withepisomal-5TF-vectors.

FIGS. 7A-7C show episomal vectors are lost from hPSC-derived-5TF cells.(FIG. 7A) Scheme depicting sample collection for ddPCR analysis. (FIG.7B) EBNA1 detection by ddPCR in DNA extracted from human CD45⁺ cellssorted from the bone marrow of primary mice transplanted with epi-5TFcells at 6 (n=5 mice), 10 (n=6 mice) and 16 (n=6 mice) weeks posttransplantation. 48 hours after transfection of HE cells withepisomal-5TF-vectors, GFP⁺ cells were sorted and used for DNA extractionto estimate the initial copy number of plasmids per genome (Epi-5TF 0w,n=3 replicates). (FIG. 7C) Plots showing representative ddPCR results at0, 6, 10 and 16 weeks. Blue dots represent double negative droplets(NN), pink dots represent single positive droplets for the referencegene (CD90) (NP), green dots represent single positive droplets for thetarget gene (EBNA1) (PN) and yellow dots indicate double positivedroplets for both target and reference genes (PP).

FIGS. 8A-8F show limiting-dilution analysis reveals HSPC frequency ofengrafted cell populations. (FIG. 8A) Schematic illustration of thelimiting dilution transplantation strategy followed to evaluate HSPCsfrequency. (FIG. 8B) Percentage of human CD45⁺ cells detected by flowcytometry in bone marrow of injected leg of secondary mice transplantedwith 30,000 (30K), 10,000 (10K) or 5,000 (5K) human CD34⁺ cells isolatedfrom bone marrow of primary transplanted mice. Engraftment wasdetermined by flow cytometry of bone marrow at 10 weeks posttransplantation (n=8 mice transplanted with 30K lenti-5TF cells, n=13mice transplanted with 30K epi-5TF cells, n=8 mice transplanted with 30Kcord blood cells; n=8 mice transplanted with 10K lenti-5TF cells, n=1mice transplanted with 10K epi-5TF cells, n=9 mice transplanted with 10Kcord blood cells; n=7 mice transplanted with 5K lenti-5TF cells, n=8mice transplanted with 5K epi-5TF cells and n=9 mice transplanted with5K cord blood cells). (FIG. 8C) Percentage of human CD45⁺ cells detectedby flow cytometry in injected (grey) and contralateral (orange) leg ofengrafted mice (those with ≥0.01% human chimerism). LS1-LS6 weretransplanted with human CD34⁺ cells derived from the bone marrow ofprimary mice transplanted with lenti-5TF cells. ES1-ES12 weretransplanted with human CD34⁺ cells derived from bone marrow of primarymice transplanted with epi-5TF. CBS1-CBS9 were transplanted with humanCD34⁺ cells derived from bone marrow of primary mice transplanted withumbilical cord blood cells. Line indicates 0.01% of human chimerism.(FIG. 8D) Lineage distribution of myeloid (M; CD33+), B (B; CD19⁺) and T(T; CD3⁺) cells within the human CD45⁺ population of injected (I) andcontralateral (C) leg's bone marrow from secondary engrafted mice(>0.01% of human chimerism). Mice with multi-lineage engraftment areindicated with an asterisk. (FIG. 8E) FACS plots showing representativeengraftment in bone marrow of a secondary mouse transplanted with humanCD34⁺ cells isolated from the bone marrow of a primary mouse injectedwith epi-5TF cells. (FIG. 8F) Graphic representing frequency of HSPCswithin human CD34⁺ cells isolated from the bone marrow of primary miceengrafted with epi-5TF cells, lenti-5TF cells or cord blood (defined as≥0.01% multi-lineage human chimerism) calculated by ELDA software(http://bioinf.wehi.edu.au/software/elda/). The bottom table indicatesthe estimate HSPC frequency and confidence intervals.

FIGS. 9A-9G show comparison of differentiated blood cells derived fromcord blood and episomal-5TF cells. (FIG. 9A) Scheme depicting samplepreparation for single-cell analysis. (FIG. 9B) Unsupervisedhierarchical clustering analysis of cord blood and episomal-5TFsingle-cell transcriptomes. (FIG. 9C) t-SNE analysis color-coded bysubpopulations identified using graph-based clustering of epi-5TFsingle-cell transcriptomes. (FIG. 9D) t-SNE analysis color-coded bysubpopulations identified using graph-based clustering of cord bloodsingle-cell transcriptomes. (FIG. 9E) Gene ontology analysis ofsubpopulation-specific gene signatures identified from epi-5TF cellssubpopulations. Immune response (IR), antigen processing andpresentation of exogenous antigen (APPEA), antigen processing andpresentation of peptide antigen (APPPA), antigen processing andpresentation of peptide or polysaccharide antigen (APPPPA),transesterification reactions with bulged adenosine as nucleophile(TRBAN), spliceosome (S), transesterification reactions (TR), sisterchromatid (SC). GO terms shared by epi-5TF and cord blood cells areindicated in bold. (FIG. 9F) Gene ontology analysis ofsubpopulation-specific gene signatures identified from cord blood cellssubpopulations. Cellular response (CR), positive regulation (PR), immuneresponse (IR), catabolic process (CP), nonsense-mediated decay (NMD),cotranslational protein targeting to membrane (CPTM), bundle assembly(BA). GO terms shared by epi-5TF and cord blood cells are indicated inbold. (FIG. 9G) Assignment of epi-5TF cells as belonging to one or morecord blood clusters based on majority vote (binary classification, 15%of the trees in the forest).

FIGS. 10A and 10B show episomal vectors and expression of 5TFs in HEcells. (FIG. 10A) Polycistronic epi-5TFs vectors used for HSPCsgeneration. (FIG. 10B) qRT-PCR analysis of HOXA9, HOXA5, RUNX1, LCOR andERG in HE cells infected or transfected with lentiviral or episomalvectors. Plot indicates relative mRNA levels to hemogenic endothelium at48h after the cell's infection or transfection.

FIGS. 11A-11E show fluorescence minus one (FMO) controls. (FIG. 11A)Lineage panel FACS plots and FMO controls. (FIG. 11B) HSPC panel FACSplot and FMO controls. (FIG. 11C) Neutrophil panel FACS plot and FMOcontrols. (FIG. 11D) T-cells panel FACS plot and FMO controls. (FIG.11E) B-cells panel FACS plot and FMO controls.

FIG. 12 shows detection of episomal vectors by FACS analysis of GFP. GFPdetection by flow cytometry within the human CD45⁺ cells identified inbone marrow of primary transplanted mice analyzed at 6 (n=5 mice), 10(n=6 mice) and 16 (n=6 mice) weeks after injection with episomal-5TFcells.

FIGS. 13A-13J show single-cell RNA-seq analysis of cord blood andepisomal-5TFs-derived cells. (FIG. 13A) Subpopulation-specific genesignatures identified from epi-5TF single-cell transcriptomes. (FIG.13B) Subpopulation-specific gene signatures identified from cord bloodsingle-cell transcriptomes. (FIG. 13C) Classification probabilities ofepi-5TF as cord blood subpopulations. (FIG. 13D) Mean classificationprobability of epi-5TF as cord blood clusters. (FIG. 13E) Subpopulationstructure identified by CellRouter to reconstruct a differentiationtrajectory from subpopulations on the extremes of the t-SNE plot (CR_8and CR_5). (FIG. 13F) Clustering of gene expression trends duringgranulocyte differentiation (transition from CR_8 to CR_5). (FIG. 13G)Gene ontology analysis of genes clustered into 5 transcriptionalprofiles along the granulocyte differentiation trajectory. Immuneresponse (IR), antigen processing and presentation (APP), peptideantigen (PA), peptide or polysaccharide antigen (PPA), exogenous peptideantigen (EPA), transmembrane transport (TT). (FIG. 13H) Top genesidentified by CellRouter as dynamically regulated during granulocytedifferentiation. (FIG. 13I) Kinetic trends of genes presented in FigureS4H downregulated during granulocyte differentiation. (FIG. 13J) Kinetictrends of genes presented in Figure S4H upregulated during granulocytedifferentiation.

FIG. 14 show limiting dilution data for calculating frequency of HSPCfrom umbilical cord blood, epi-5TF and lenti-5TF-derived cells. Dose isthe number of transplanted cells, tested is the number of mice analyzedand response is the number of mice that showed multi-lineage engraftment(human bone marrow chimerism ≥0.01%).

FIG. 15A shows schematic of epicomal vector used herein.

FIG. 15B shows FACS plot of GFP and BNA-positive cells. Expressiondriven by episomal vector of FIG. 15A.

FIG. 16 shows FACS plot of cell expressing the indicated markers. NSGW41mice were intrafemorally injected with transfected cells the followingday after the cell's transfection (Injection of 100,000 cells per mice).8 weeks after the cells transplantation is was possible to detect humancells in peripheral blood of mice injected with episomal 5F cells.

FIGS. 17A and 17B show cells expressing human CD45 in bone marrow afterinjection. Injected mice were sacrificed 10-12 weeks after the cell'sinjection and their bone marrow was analyzed for the presence of humancells (hCD45, FIG. 17A) detecting six mice that showed human cells intheir bone marrow. Multi-lineage capacity of the engrafted cells wasalso analyzed (FIG. 17B), revealing four mice with multi-lineage (bothlynphoid and myeloid cells).

FIGS. 18A and 18B show lentiviral (L2 and L6) and episomal (E3, E5 andE6) injected mice with remarkable engraftment. FIG. 18A is a FACs plotsof hCD45+ cells found in these mice bone marrow 10-12 weeks after thecell's injection. FIG. 18B is a FACS plot of multi-lineage FACs analysisof human engrafted cells.

FIGS. 19A and 19B show long-term capacity of episomal 5F-transfectedcells was evaluated sacrifying mice 16 weeks after the cell'sintrafemoral injection. FIG. 19A shows % of hCD45 cells found in micebone marrow. This experiment confirmed the long-term potential ofepisomal 5F cells. FIG. 19B shows cells derived from episomal 5F cellsalso showed multi-lineage capacity.

FIG. 20 shows STR analysis for identifying short tandem repeats was doneusing DNA extracted from episomal 5F-derived engrafted cells. Cells wererecovered from mice's bone marrow, 16 weeks after the cell'stransplantation. This analysis confirmed the induced pluripotent stemcell origin of the episomal 5F-derived cells.

FIGS. 21A-21C show detection of episomal plasmid in engrafted cells.FIG. 21A is a schematic of the episomal plasmid. qPCR (FIG. 21B) anddroplet digital PCR analysis (FIG. 21C) of DNA extracted from humanengrafted cells derived from episomal 5F cells confirmed that theepisomal plasmids are lost from the cells 16 weeks after the cell'stransplantation.

FIGS. 22A and 22B show detection of lentiviral plasmid in engraftedcells. FIG. 22A is a schematic of the lentiviral plasmid. No lentiviralvectors were detected on the episomal engrafted cells, confirming thatthere was no cross-contamination between samples.

FIG. 23A-23C show engraftment of episomal 5F-cells in bone marrow. FIG.23A is a schematic of the experiment. FIG. 23B shows percentage of humanCD45+ cells found in mice's bone marrow analyzed between 10-12 weeksafter lentiviral or episomal 5TF-cell's transplantation. Mice injectedwith human cord-blood cells were used as control. FIG. 23C showsrepresentative FACs plots of lentiviral (L2, L6, L8) and episomal (E3,E5, E6) 5TF-injected mice with remarkable engraftment of human cells inbone marrow.

FIGS. 24A and 24B show episomal 5F-cells have long term engraftmentpotential and homing capacity. Long-term engraftment analysis of human5TF-cells. FIG. 24A show percentage of human CD45+ cells found in mice'sbone marrow analyzed at 16 weeks after lentiviral or episomal 5TF-cell'stransplantation. In grey, data from injected leg (I) and in green humanCD45+ cells found at the contralateral leg (CL) FIG. 24B showmulti-lineage contribution of human cells in bone marrow of engraftedmice analyzed 16 weeks after cell's intrafemoral injection. Bone marrowwas analyzed for human myeloid cells (M; CD33+), B cells (B; CD19+), andT cells (T; CD3+). Mice with multi-lineage engraftment are indicatedwith a black dot. Mice injected with human cord-blood cells were used ascontrol.

DETAILED DESCRIPTION

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. It should beunderstood that this disclosure is not limited to the particularmethodology, protocols, and reagents, etc., described herein and as suchcan vary. The terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe embodiments of the present disclosure, which is defined solely bythe claims.

Definitions of common terms in molecular biology can be found in TheMerck Manual of Diagnosis and Therapy, 19th Edition, published by MerckSharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3), (2015 digital onlineedition at the website of Merck Manuals), Robert S. Porter et al.(eds.), The Encyclopedia of Molecular Cell Biology and MolecularMedicine, published by Blackwell Science Ltd., 1999-2012 (ISBN9783527600908); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by WernerLuttmann, published by Elsevier, 2006; Janeway's Immunobiology, KennethMurphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014(ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones &Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green andJoseph Sambrook, Molecular Cloning: A Laboratory Manual, 4^(th) ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA(2012) (ISBN 1936113414); Davis et al., Basic Methods in MolecularBiology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.)Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology(CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN047150338X, 9780471503385), Current Protocols in Protein Science (CPPS),John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and CurrentProtocols in Immunology (CPI) (John E. Coligan, A D A M Kruisbeek, DavidH Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons,Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which areall incorporated by reference herein in their entireties. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

Unless otherwise stated, the embodiments of the present disclosure wereperformed using standard procedures known to one skilled in the art, forexample, in Michael R. Green and Joseph Sambrook, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., USA (2012); Davis et al., Basic Methods in MolecularBiology, Elsevier Science Publishing, Inc., New York, USA (1986);Current Protocols in Molecular Biology (CPMB) (Fred M. Ausubel, et al.ed., John Wiley and Sons, Inc.), Current Protocols in Immunology (CPI)(John E. Coligan, et. al., ed. John Wiley and Sons, Inc.), CurrentProtocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., JohnWiley and Sons, Inc.), Culture of Animal Cells: A Manual of BasicTechnique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005),Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P.Mather and David Barnes editors, Academic Press, 1st edition, 1998),Methods in Molecular biology, Vol. 180, Transgenesis Techniques by AlanR. Clark editor, second edition, 2002, Humana Press, and Methods inMeolcular Biology, Vo. 203, 2003, Transgenic Mouse, editored by MartenH. Hofker and Jan van Deursen, which are all herein incorporated byreference in their entireties.

It should be understood that embodiments of the present disclosure arenot limited to the particular methodology, protocols, and reagents,etc., described herein and as such may vary. The terminology used hereinis for the purpose of describing particular embodiments only, and is notintended to limit the scope of the embodiments of the presentdisclosure, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages willmean ±1%.

All patents and publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the embodiments of the present disclosure. Thesepublications are provided solely for their disclosure prior to thefiling date of the present application. Nothing in this regard should beconstrued as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior embodiments of the presentdisclosure or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The disclosure described herein, in a preferred embodiment, does notconcern a process for cloning human beings, processes for modifying thegerm line genetic identity of human beings, uses of human embryos forindustrial or commercial purposes or processes for modifying the geneticidentity of animals which are likely to cause them suffering without anysubstantial medical benefit to man or animal, and also animals resultingfrom such processes.

The disclosure described herein does not concern the destruction of ahuman embryo.

There is no one well tested and reliable method of producing HSCs andHSPCs from PSCs where these HSCs and HSPCs have all the hematopoieticlineage potentials and would engraft and reconstitute in vivo. i.e.,multi-lineage HSCs and HSPCs that capable of producing blood cells invivo. The disclosure seeks to provide improved PSCs-derived HSCs andHSPCs that exhibit long-term multilineage hematopoiesis in vivo afterimplantation in a host subject. For example, long-term multilineagehematopoiesis in vivo for at least 12 weeks or longer afterimplantation.

A variety of tissue lineages can be derived in vitro by stepwiseexposure of pluripotent stem cells (PSCs) to morphogens in an attempt tomimic embryonic development¹, or by conversion of one differentiatedcell type directly into another by enforced expression of mastertranscription factors (TFs)².

Despite considerable effort, neither approach has yielded functionalhuman hematopoietic stem cells (HSCs). Building upon recent evidencethat HSCs derive from definitive hemogenic endothelium (HE)³⁻⁹, theinventors performed morphogen-directed differentiation of human PSCsinto HE followed by combinatorial screening of 26 candidateHSC-specifying TFs for the potential to promote hematopoieticengraftment in irradiated immune deficient murine hosts. The inventorsrecovered seven TFs (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, SPI1) thattogether were sufficient to convert HE into hematopoietic stem andprogenitor cells (HSPCs) that engraft primary and secondary murinerecipients with myeloid cells, beta globin-expressing erythrocytes,IgM+/CD19+ B-cells, and αβ and γδ T-cells. Five TFs, ERG, HOXA5, HOXA9,LCOR, and RUNX1, are the minimum TFs necessary to convert HE into HSPCs.Integration analysis of virally-transduced transgenes detected commonclones in myeloid and lymphoid lineages, indicating derivation ofHSC-like cells from PSCs. This combined approach of morphogen-drivendifferentiation and TF-mediated cell fate conversion from PSCs yieldsHSPCs that hold promise for modeling hematopoietic disease in humanizedmice and for therapeutic strategies in genetic blood disorders.

Described herein, the inventors demonstrated a process of makingPSCs-derived HSCs and HSPCs that would differentiate into all thehematopoietic lineage potentials and would also engraft well in the hostafter transplantation so that there is sufficient engrafted cells tosustain blood production in vivo. This method produces functionallyrelevant HSCs and HSPCs in sufficient quantities for both meaningfulexperimental and therapeutic purposes. For example, in vitroexperiments, these PSCs-derived HSCs and HSPCs can be differentiated tothe desired hematopoietic lineage, e.g., erythroid cells, lymphoidcells, and myeloid cells, for further studies. For example, in in vivostudies, these PSCs-derived HSCs and HSPCs would engraft in the host,and differentiate into the variety of hematopoietic progeny cells, andreconstitute and populate the circulatory and immune system of the host.

Embodiments of the present disclosure are based, in part, to thediscovery of that transcription factors, ERG, HOXA5, HOXA9, LCOR, RUNX1,HOXA10, and SPI1, would bring about the differentiation of HSCs andHSPCs from PSC-derived hemogenic endothelia cells (HE). First, theinventors showed that embryonic bodies (EB) are made from pluripotentstem cells, e.g., including induced pluripotent cells. Second, the HEare harvested from the EB. Then, the HE cells are induced to undergo EHTand subsequently transfected with exogenous copies of at least thefollowing transcription factors: ERG, HOXA9, HOXA5, LCOR and RUNX1, topromote differentiation of the HE into HSCs and HSPCs that exhibit allthe hematopoietic lineage potentials. These multi-lineage HSCs and HSPCsengraft in recipient host after implantation and made all kinds of bloodcells in vivo after implantation.

Accordingly, in one aspect, provided herein is a method for making HSCsand HSPCs comprising in vitro transfecting hemogenic endothelia cells(HE) with an exogenous gene coding copy of each of the followingtranscription factors ERG, HOXA9, HOXA5, LCOR and RUNX1, wherein thetranscription factors are expressed in the transfected cells to producea population of multilineage HSCs and HSPCs that engrafts in recipienthost after implantation.

In another aspect, this disclosure provides is a method of making HSCsand HSPCs comprising (a) generating EB from PSCs; (b) isolatinghemogenic endothelia cells (HE) from the resultant population of EB; (c)inducing EHT in culture in the isolated HE in order to obtainhematopoietic stem cells, and (d) in vitro transfecting the induced HEwith an exogenous gene coding copy of each of the followingtranscription factors ERG, HOXA9, HOXA5, LCOR and RUNX1, wherein thetranscription factors are expressed in the transfected cells to producea population of multilineage HSCs and HSPCs. In one embodiment, themethod further comprises selecting EBs that are generated from the PSCs,prior to isolating the HE. In one embodiment, the HE is isolated fromthe selected EBs.

In some aspects, this disclosure provides methods for enhancing orimproving the in vivo engraftment, or reconstitution, or both ofhematopoietic related cells that have been implanted into a subject. Inone embodiment, the method comprises providing populations ofmultilineage HSCs and HSPCs that have an exogenous gene coding copy ofeach of the following transcription factors ERG, HOXA9, HOXA5, LCOR andRUNX1, and optionally an exogenous gene coding copy of the transcriptionfactors HOXA10, and SPI1, and implanting into a host subject. In oneembodiment, the multilineage HSCs and HSPCs further comprise and anexogenous gene coding copy of each of the following reprogrammingfactors OCT4, SOX2, KLF4 and optionally c-MYC or nanog and LIN28. Inanother embodiment, the method comprises making HSCs and HSPCs by anyone method in this disclosure and implanting into a host subject. In oneembodiment, the subject donates some mature cells from which iPSCs areinduced. From the iPSCs, EBs are induced from which HEs are isolated andinduced to EHT, followed by the transfection of transcription factorgenes to produce HSCs and HSPCs. These HSCs and HSPCs are then implantedback into the donor subject, wherein the donor and recipient is thesubject. In one embodiment, a donor subject donates some mature cellsfrom which iPSCs are induced. From the iPSCs, EBs are induced from whichHEs are isolated and induced to EHT, followed by the transfection oftranscription factor genes to produce HSCs and HSPCs. These HSCs andHSPCs are then implanted back into a recipient subject, wherein thedonor and recipient are two different subjects.

In some aspects, this disclosure provides compositions of modified (alsoreferred to as engineered) cells for use in in vivo cellular replacementtherapy, for the manufacture of medicament for treatment ofhematological diseases, blood disorders, hematopoietic disorders, andfor in vitro studies of disease modeling, drug screening, andhematological diseases. In one embodiment, the engineered cells aremultilineage HSCs and HSPCs that have an exogenous gene coding copy ofeach of the following transcription factors ERG, HOXA9, HOXA5, LCOR andRUNX1, and optionally also contain an exogenous gene coding copy of thetranscription factors HOXA10, and SPI1. In one embodiment, theengineered cells are multilineage HSCs and HSPCs that further comprisean exogenous gene coding copy of each of the following reprogrammingfactors OCT4, SOX2, KLF4 and optionally c-MYC or NANOG and LIN28. Inanother embodiment, the engineered cells are HSCs and HSPCs made by anyone method described in this disclosure. In one embodiment, theengineered cells are CD34+. In another embodiment, the engineered cellsare CD34+ and CD45+. In another embodiment, the engineered cells areCD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is an engineered cellderived from a population of HE that is produced by a method comprising(a) generating EB from PSCs; (b) isolating HE from the resultantpopulation of EB; (c) inducing EHT in culture in the isolated HE inorder to obtain hematopoietic stem cells, and (d) in vitro transfectingthe population of HE with an exogenous gene coding copy of each of thefollowing transcription factors ERG, HOXA9, HOXA5, LCOR and RUNX1. Inone embodiment, the method further comprises selecting EBs that aregenerated from the PSCs, prior to isolating the HE. In one embodiment,the HE is isolated from the selected EBs. In another embodiment, thepopulation of HE is further transfected with an exogenous gene codingcopy of the transcription factors HOXA10, and SPI1. In one embodiment,the engineered cells are the multilineage HSCs and HSPCs are producedfrom the resultant transfection of the described TFs. In one embodiment,the engineered cell further comprise an exogenous gene coding copy ofeach of the following reprogramming factors OCT4, SOX2, KLF4 andoptionally c-MYC or NANOG and LIN28. In one embodiment, the engineeredcells are CD34+. In another embodiment, the engineered cells are CD34+and CD45+. In another embodiment, the engineered cells are CD34+, CD45+,and CD38 negative.

In another aspect, this disclosure provides is an engineered cellderived from a population of HE that is produced by a method comprisingin vitro transfecting the population of HE with an exogenous gene codingcopy of each of the following transcription factors ERG, HOXA9, HOXA5,LCOR and RUNX1. In another embodiment, the population of HE is furthertransfected with an exogenous gene coding copy of the transcriptionfactors HOXA10, and SPI1. In one embodiment, the engineered cells arethe multilineage HSCs and HSPCs are produced from the resultanttransfection of the described TFs. In one embodiment, the engineeredcell further comprise an exogenous gene coding copy of each of thefollowing reprogramming factors OCT4, SOX2, KLF4 and optionally c-MYC orNANOG and LIN28. In one embodiment, the engineered cells are CD34+. Inanother embodiment, the engineered cells are CD34+ and CD45+. In anotherembodiment, the engineered cells are CD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is an engineered cellcomprises an exogenous copy of each of the following transcriptionfactors ERG, HOXA9, HOXA5, LCOR and RUNX1. In another embodiment, theengineered cell further comprises an exogenous gene coding copy of thetranscription factors HOXA10, and SPI1. In one embodiment, theengineered cell further comprise an exogenous gene coding copy of eachof the following reprogramming factors OCT4, SOX2, KLF4 and optionallyc-MYC or NANOG and LIN28. In another embodiment, the engineered cellsare HSCs and HSPCs made by any one method described in this disclosure.In one embodiment, the engineered cells are CD34+. In anotherembodiment, the engineered cells are CD34+ and CD45+. In anotherembodiment, the engineered cells are CD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is a composition comprisinga population of engineered cells derived from a population of HE andproduced by a method comprising (a) generating embryonic bodies (EB)from pluripotent stem cells; (b) isolating hemogenic endothelia cells(HE) from the resultant population of EB; (c) inducing EHT in culture inthe isolated HE in order to obtain hematopoietic stem cells, and (d) invitro transfecting the population of HE with an exogenous gene codingcopy of each of the following transcription factors ERG, HOXA9, HOXA5,LCOR and RUNX1. In one embodiment, the population of HE is furthertransfected with an exogenous gene coding copy of the transcriptionfactors HOXA10, and SPI1. In one embodiment, the engineered cells arethe multilineage HSCs and HSPCs are produced from the resultanttransfection of the described TFs. In one embodiment, the engineeredcell further comprise an exogenous gene coding copy of each of thefollowing reprogramming factors OCT4, SOX2, KLF4 and optionally c-MYC orNANOG and LIN28. In one embodiment, the engineered cells are CD34+. Inanother embodiment, the engineered cells are CD34+ and CD45+. In anotherembodiment, the engineered cells are CD34+, CD45+, and CD38 negative. Insome embodiments, this composition is useful for cellular replacementtherapy in a subject.

In another aspect, this disclosure provides is a composition comprisinga population of engineered cells derived from a population of HE andproduced by a method comprising in vitro transfecting the population ofHE with an exogenous gene coding copy of each of the followingtranscription factors ERG, HOXA9, HOXA5, LCOR and RUNX1. In oneembodiment, the population of HE is further transfected with anexogenous gene coding copy of the transcription factors HOXA10, andSPI1. In another embodiment, the engineered cells are HSCs and HSPCsmade by any one method described in this disclosure. In one embodiment,the engineered cells are CD34+. In another embodiment, the engineeredcells are CD34+ and CD45+. In another embodiment, the engineered cellsare CD34+, CD45+, and CD38 negative. In some embodiments, thiscomposition is useful for cellular replacement therapy in a subject, andfor in vitro studies of disease modeling, drug screening, andhematological diseases.

In another aspect, this disclosure provides is a composition comprisinga population of engineered cells wherein the cells comprise an exogenousgene coding copy of each of the following transcription factors ERG,HOXA9, HOXA5, LCOR and RUNX1. In one embodiment, the cells furthercomprise an exogenous gene coding copy of the transcription factorsHOXA10, and SPI1. In another embodiment, the engineered cells are HSCsand HSPCs made by any one method described in this disclosure. In oneembodiment, the engineered cells are CD34+. In another embodiment, theengineered cells are CD34+ and CD45+. In another embodiment, theengineered cells are CD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is a pharmaceuticalcomposition comprising a population of engineered cells derived from apopulation of HE and a pharmaceutically acceptable carrier, wherein theengineered cells are produced by a method comprising (a) generating EBfrom PSCs; (b) isolating HE from the resultant population of EB; (c)inducing EHT in culture in the isolated HE in order to obtainhematopoietic stem cells, and (d) in vitro transfecting the populationof HE with an exogenous gene coding copy of each of the followingtranscription factors ERG, HOXA9, HOXA5, LCOR and RUNX1. In oneembodiment, the population of HE is further transfected with anexogenous gene coding copy of the transcription factors HOXA10, andSPI1. In one embodiment, the engineered cells are CD34+. In anotherembodiment, the engineered cells are CD34+ and CD45+. In anotherembodiment, the engineered cells are CD34+, CD45+, and CD38 negative. Insome embodiments, this pharmaceutical composition is useful for cellularreplacement therapy in a subject.

In another aspect, this disclosure provides is a pharmaceuticalcomposition comprising a population of engineered cells derived from apopulation of HE and a pharmaceutically acceptable carrier, wherein theengineered cells are produced by a method comprising in vitrotransfecting the population of HE with an exogenous gene coding copy ofeach of the following transcription factors ERG, HOXA9, HOXA5, LCOR andRUNX1. In one embodiment, the population of HE is further transfectedwith an exogenous gene coding copy of the transcription factors HOXA10,and SPI1. In one embodiment, the engineered cells are CD34+. In anotherembodiment, the engineered cells are CD34+ and CD45+. In anotherembodiment, the engineered cells are CD34+, CD45+, and CD38 negative. Insome embodiments, this pharmaceutical composition is useful for cellularreplacement therapy in a subject.

In another aspect, this disclosure provides is a pharmaceuticalcomposition comprising a population of engineered cells and apharmaceutically acceptable carrier, wherein the engineered cellscomprise an exogenous gene coding copy of each of the followingtranscription factors ERG, HOXA9, HOXA5, LCOR and RUNX1. In oneembodiment, the engineered cells further comprise an exogenous genecoding copy of the transcription factors HOXA10, and SPI1. In oneembodiment, the engineered cells are CD34+. In another embodiment, theengineered cells are CD34+ and CD45+. In another embodiment, theengineered cells are CD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is a method of cellularreplacement therapy in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of a population ofengineered cells to a recipient subject, the population of engineeredcells are produced by a method comprising (a) generating EB from PSCs;(b) isolating HE from the resultant population of EB; (c) inducing ENTin culture in the isolated HE in order to obtain hematopoietic stemcells, and (d) in vitro transfecting the population of HE with anexogenous gene coding copy of each of the following transcriptionfactors ERG, HOXA9, HOXA5, LCOR and RUNX1. In one embodiment, thepopulation of HE is further transfected with an exogenous gene codingcopy of the transcription factors HOXA10, and SPI1. In one embodiment,the engineered cells are CD34+. In another embodiment, the engineeredcells are CD34+ and CD45+. In another embodiment, the engineered cellsare CD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is a method of cellularreplacement therapy in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of a population ofengineered cells to a recipient subject, the population of engineeredcells are produced by a method comprising in vitro transfecting thepopulation of HE with an exogenous gene coding copy of each of thefollowing transcription factors ERG, HOXA9, HOXA5, LCOR and RUNX1. Inone embodiment, the population of HE is further transfected with anexogenous gene coding copy of the transcription factors HOXA10, andSPI1. In one embodiment, the engineered cells are CD34+. In anotherembodiment, the engineered cells are CD34+ and CD45+. In anotherembodiment, the engineered cells are CD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is a method of cellularreplacement therapy in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of a population ofengineered cells to a recipient subject, the population of engineeredcells comprise an exogenous gene coding copy of each of the followingtranscription factors ERG, HOXA9, HOXA5, LCOR and RUNX1. In oneembodiment, the engineered cells further comprise an exogenous genecoding copy of the transcription factors HOXA10, and SPI1. In oneembodiment, the engineered cells are CD34+. In another embodiment, theengineered cells are CD34+ and CD45+. In another embodiment, theengineered cells are CD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides engineered cells derivedfrom a population of HE and produced by a method described herein. Inone embodiment, the engineered cells are CD34+. In another embodiment,the engineered cells are CD34+ and CD45+. In another embodiment, theengineered cells are CD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is a composition comprisinga population of engineered cells described herein. In one embodiment,the engineered cells are multilineage HSCs and HSPCs are produced fromthe resultant transfection of the described TFs. In one embodiment, theengineered cells are CD34+. In another embodiment, the engineered cellsare CD34+ and CD45+. In another embodiment, the engineered cells areCD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is a pharmaceuticalcomposition comprising a population of engineered cells described hereinand a pharmaceutically acceptable carrier. In one embodiment, theengineered cells are CD34+. In another embodiment, the engineered cellsare CD34+ and CD45+. In another embodiment, the engineered cells areCD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is a pharmaceuticalcomposition described herein for use in cellular replacement therapy ina subject.

In another aspect, this disclosure provides is a method of cellularreplacement therapy in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of a population ofengineered cells described, or a composition described, or apharmaceutical composition described to a recipient subject.

In one embodiment of any one aspect described, the method of generatingHSCs and HSPCs described is an in vitro or ex vivo method.

In one embodiment of any one method, engineered cell, or compositiondescribed, the multilineage hematopoietic progenitor cells are generatedby introducing in vitro or ex vivo each of the following transcriptionfactors ERG, HOXA9, HOXA5, LCOR and RUNX1, in the HE cells derived fromPSC-induced EBs. For example, by transfecting with a vector or more, thevector(s) collectively carry an exogenous gene coding copy of each ofthe following transcription factors, ERG, HOXA9, HOXA5, LCOR and RUNX1,for in vivo expression of the transcription factor in the transfectedcells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the multilineage hematopoietic progenitor cells are generatedby contacting a population of HEs with a vector or more, wherein thevector(s) collectively carrying an exogenous gene coding copy of each ofthe following transcription factors, ERG, HOXA9, HOXA5, LCOR and RUNX1,for the in vivo expression of the factors in the contacted cells, andwherein the transfected transcription factors are expressed in vivo inthe contacted cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the method further comprising in vitro transfecting the HEwith an exogenous gene coding copy of the transcription factor, HOXA10,wherein the transfected transcription factor is expressed in vivo in thetransfected cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the method further comprising in vitro transfecting the HEwith an exogenous gene coding copy of the transcription factor, SPI1,wherein the transfected transcription factor is expressed in vivo in thetransfected cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the method further comprising in vitro transfecting the HEwith an exogenous gene coding copy of the transcription factors, HOXA10and SPI1, wherein the transfected transcription factors are expressed invivo in the transfected cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cell of this disclosure is a mammalian cell.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered mammalian cell is a primate cell.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered primate cell is a human cell.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cell is CD34+.

In another embodiment of any one method, engineered cell, or compositiondescribed, the engineered cell is CD34+ and CD45+.

In another embodiment of any one method, engineered cell, or compositiondescribed, the engineered cell is CD34+, CD45+, and CD38 negative.

In another embodiment of any one method, engineered cell, or compositiondescribed, the engineered cell has an exogenous gene of at least one ofthe following transcription factors: ERG, HOXA9, HOXA5, HEXA10, SPI1,LCOR and RUNX1.

In another embodiment of any one method, engineered cell, or compositiondescribed, the engineered cell has an exogenous gene of at least one ofthe following reprogramming factors: OCT4, SOX2, KLF4, c-MYC, NANOG, andLIN28.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cells disclosed herein are multilineage HSCsand HSPCs that engraft in vivo in a host recipient subject and produceblood cells in vivo.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cells disclosed herein are multilineage HSCsand HSPCs that reconstitutes the hematopoietic system in vivo whentransplanted into a host recipient subject.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cells disclosed herein are multilineage HSCsand HSPCs that differentiate to myeloid cells in vivo, and the myeloidcells produce MPO upon PMA or cytokine stimulation in vivo.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cells disclosed herein are multilineage HSCsand HSPCs that differentiate to functional T- and B-cells in vivo, thefunctional T- and B-cells produce IgM and IgG. The functional T- andB-cells also undergo immunoglobulin class switching in response toovalbumin stimulation. The functional T- and B-cells also producesINF-γ.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cells produce blood cells in vivo whenengrafted in vivo in a host recipient subject.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cells reconstitutes the hematopoietic systemin vivo when transplanted into a host recipient subject.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cells differentiate to myeloid cells in vivo,and the myeloid cells produce MPO upon PMA or cytokine stimulation invivo.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cells differentiate to functional immune cellsin vivo, e.g., T- and B-cells, wherein the functional immune cellsproduce IgM and IgG. The functional immune cells also undergoimmunoglobulin class switching in response to ovalbumin stimulation. Thefunctional immune cells also produce INF-γ.

In one embodiment of any one method, engineered cell, or compositiondescribed, the HE cells are cells that are derived from embryoid bodies(EBs) obtained from a population of pluripotent stem cells (PSC). In oneembodiment, the HE are definitive HE.

In one embodiment of any one method, engineered cell, or compositiondescribed, the population of PSC is induced pluripotent stem cells(iPSc) or embryonic stem cells (ESC).

In one embodiment of any one method, engineered cell, or compositiondescribed, the PSC are human PSC or mouse PSC.

In one embodiment of any one method, engineered cell, or compositiondescribed, the iPSCs are produced by in vitro or ex vivo introducingexogenous copies of only three reprogramming factors OCT4, SOX2, andKLF4 into mature cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the iPSC having exogenous copies of OCT4, SOX2, and KLF4 isfurther introduced in vitro or ex vivo with an exogenous copy of c-MYCinto the cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the iPSC having exogenous copies of OCT4, SOX2, and KLF4 isfurther introduced in vitro or ex vivo with an exogenous copies of NANOGand LIN28 into the cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the iPSC are produced by introducing in vitro or ex vivoexogenous copies of reprogramming factors OCT4, SOX2, and KLF4, andoptionally with c-MYC or NANOG and LIN28 into the mature cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the iPSC are produced by in vitro or ex vivo contacting themature cells with a vector or more, wherein the vector(s) collectivelycarry exogenous copies of reprogramming factors OCT4, SOX2, and KLF4,and optionally with c-MYC or NANOG and LIN28 into mature cells, andwherein the reprogramming factors are expressed in vivo in the contactedmature cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the mature cells from which iPSC are made can be from anycell type in a donor subject. For examples, cells from a blood sample,or bone marrow sample, B lymphocytes (B-cells), T lymphocytes,(T-cells), fibroblasts, keratinocytes etc. In some embodiments, anymature cell type from the donor subject can be used except a cell withno nucleus, such as a human red blood cell.

In one embodiment of any one method, engineered cell, or compositiondescribed, the iPSC are produced by in vitro or ex vivo introducing thedisclosed reprogramming factors two or more times into the mature cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the iPSC are produced by in vitro or ex vivo contacting themature cells with the disclosed vector(s) factors two or more times intothe mature cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the mature cells from which iPSC are made are mammaliancells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the mature cells from which iPSC are made are primate cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the mature cells from which iPSC are made are human cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the mature cells from which iPSC are made are autologous to arecipient subject who would be receiving the engineered cells that arederived from the mature cells according to any one of the methodsdescribed in this disclosure.

In one embodiment of any one method, engineered cell, or compositiondescribed, the mature cells from which iPSC are made are HLA matchedwith a recipient subject who would be receiving the engineered cellsthat are derived from the mature cells according to any one of themethods described in this disclosure.

In one embodiment of any one engineered cell or composition described,the pharmacological acceptable carrier is not cell culture media.

In one embodiment of any one composition described, the pharmacologicalcomposition is a cryopreserved composition comprising at least onecryopreservative agent known in the art. For examples, dimethylsulphoxide (DMSO), polyvinylpyrrolidone (PVP), fetal calf serum (FCS),polyethylene glycol (PEG), glycerol, ethylene glycol (EG) and trehalose.Combinations of two or more cryopreservative agents can be used.

Pluripotent Stem Cells for Generating Embryonic Bodies, HE, and HSC andHSPC.

Pluripotent stem cells (PSCs) have the potential to give rise to all thesomatic tissues. Directed differentiation of PSCs aims to recapitulateembryonic development to generate patient-matched tissues by specifyingthe three germ layers. A common theme in directed differentiation acrossall germ layers is the propensity of PSCs to give rise to embryonic- andfetal-like cell types, which poses a problem for integration andfunction in an adult recipient. This distinction is particularlystriking in the hematopoietic system, which emerges in temporally andspatially separated waves at during ontogeny (Dzierzak and Speck, 2008).The earliest “primitive” progenitors emerge in the yolk sac at 8.5 dpcand give rise to a limited repertoire of macrophages, megakaryocytes andnucleated erythrocytes (Baron et al 2005, Tavian and Peault 2005,Ferkowicz et al 2005). These early embryonic-like progenitors aregenerally myeloid-based and cannot functionally repopulate the bonemarrow of adult recipients. By contrast, “definitive” cells withhematopoietic stem cell (HSC) potential emerge later in arterialendothelium within the aorta-gonad-mesonephros (AGM) and otheranatomical sites (Dzierzak and Speck, 2008). Directed differentiation ofPSCs gives rise to hematopoietic progenitors, which resemble those foundin the yolk sac of the early embryo. These lack functionalreconstitution potential, are biased to myeloid lineages, and expressembryonic globins.

In one embodiment of any one aspect described, the population of PSCused for generating EBs is induced pluripotent stem cells (iPS cells) orembryonic stem cells (ESC).

In one embodiment of any one aspect described, the iPS cells areproduced by introducing only reprogramming factors OCT4, SOX2, KLF4 andoptionally c-MYC or NANOG and LIN28 into mature cells.

In one embodiment of any one aspect described, the mature cells forproducing iPS cells are selected from the group consisting of Blymphocytes (B-cells), T lymphocytes, (T-cells), fibroblasts, andkeratinocytes.

In one embodiment of any one aspect described, the iPSCs are produced byintroducing the reprogramming factors two or more times into the maturecells.

Induced Pluripotent Stem Cells

In some embodiments, the pluripotent stem cells (PSCs) described hereinare derived from isolated induced pluripotent stem cells (iPSCs). Anadvantage of using iPSCs is that the cells can be derived from the samesubject to which the eventual immune cells would be reintroduced. Thatis, a somatic cell can be obtained from a subject, reprogrammed to aninduced pluripotent stem cell, and then transfected and differentiatedinto a modified immune cell to be administered to the subject (e.g.,autologous cells). Since the progenitors are essentially derived from anautologous source, the risk of engraftment rejection or allergicresponses is reduced compared to the use of cells from another subjector group of subjects. In some embodiments, the cells for generatingiPSCs are derived from non-autologous sources. In addition, the use ofiPSCs negates the need for cells obtained from an embryonic source.Thus, in one embodiment, the PSCs used in the disclosed methods are notembryonic stem cells.

Although differentiation is generally irreversible under physiologicalcontexts, several methods have been recently developed to reprogramsomatic cells to induced pluripotent stem cells. Exemplary methods areknown to those of skill in the art and are described briefly hereinbelow.

As used herein, the term “reprogramming” refers to a process that altersor reverses the differentiation state of a differentiated cell (e.g., asomatic cell). Stated another way, reprogramming refers to a process ofdriving the differentiation of a cell backwards to a moreundifferentiated or more primitive type of cell. It should be noted thatplacing many primary cells in culture can lead to some loss of fullydifferentiated characteristics. Thus, simply culturing such cellsincluded in the term differentiated cells does not render these cellsnon-differentiated cells (e.g., undifferentiated cells) or pluripotentcells. The transition of a differentiated cell to pluripotency requiresa reprogramming stimulus beyond the stimuli that lead to partial loss ofdifferentiated character in culture. Reprogrammed cells also have thecharacteristic of the capacity of extended passaging without loss ofgrowth potential, relative to primary cell parents, which generally havecapacity for only a limited number of divisions in culture.

The cell to be reprogrammed can be either partially or terminallydifferentiated prior to reprogramming. In some embodiments,reprogramming encompasses complete reversion of the differentiationstate of a differentiated cell (e.g., a somatic cell) to a pluripotentstate or a multipotent state. In some embodiments, reprogrammingencompasses complete or partial reversion of the differentiation stateof a differentiated cell (e.g., a somatic cell) to an undifferentiatedcell (e.g., an embryonic-like cell). Reprogramming can result inexpression of particular genes by the cells, the expression of whichfurther contributes to reprogramming. In certain embodiments describedherein, reprogramming of a differentiated cell (e.g., a somatic cell)causes the differentiated cell to assume an undifferentiated state(e.g., is an undifferentiated cell). The resulting cells are referred toas “reprogrammed cells,” or “induced pluripotent stem cells (iPSCs oriPS cells).”

Reprogramming can involve alteration, e.g., reversal, of at least someof the heritable patterns of nucleic acid modification (e.g.,methylation), chromatin condensation, and epigenetic changes, genomicimprinting, etc., that occur during cellular differentiation.Reprogramming is distinct from simply maintaining the existingundifferentiated state of a cell that is already pluripotent ormaintaining the existing less than fully differentiated state of a cellthat is already a multipotent cell (e.g., a common myeloid stem cell).Reprogramming is also distinct from promoting the self-renewal orproliferation of cells that are already pluripotent or multipotent,although the compositions and methods described herein can also be ofuse for such purposes, in some embodiments.

The specific approach or method used to generate pluripotent stem cellsfrom somatic cells (broadly referred to as “reprogramming”) is notcritical to the claimed embodiments of the present disclosure. Thus, anymethod that re-programs a somatic cell to the pluripotent phenotypewould be appropriate for use in the methods described herein.

Reprogramming methodologies for generating pluripotent cells usingdefined combinations of transcription factors have been described toinduced pluripotent stem cells from somatic cells. Yamanaka andTakahashi converted mouse somatic cells to ES cell-like cells withexpanded developmental potential by the direct transduction of Oct4,Sox2, Klf4, and optionally c-Myc. See U.S. Pat. Nos. 8,058,065 and9,045,738 to Yamanaka and Takahashi. iPSCs resemble ES cells as theyrestore the pluripotency-associated transcriptional circuitry and muchof the epigenetic landscape. In addition, mouse iPSCs satisfy all thestandard assays for pluripotency: specifically, in vitro differentiationinto cell types of the three germ layers, teratoma formation,contribution to chimeras, germline transmission, and tetraploidcomplementation.

Subsequent studies have shown that human iPS cells can be obtained usingsimilar transduction methods, and the transcription factor trio, OCT4,SOX2, and NANOG, has been established as the core set of transcriptionfactors that govern pluripotency. The production of iPS cells can beachieved by the introduction of nucleic acid sequences encoding stemcell-associated genes into an adult, somatic cell, using viral vectors.In general, retroviruse- or adenoviruse-based vectors are used todeliver the desired gene/nucleic acid. Other viruses used as vectorsinclude adeno-associated viruses, lentiviruses, pox viruses,alphaviruses, and herpes viruses. Additionally, non-integrative episomalvectors (oriP/EBNA-1 [Epstein Barr nuclear antigen-1], the non-viralepisomal vector pEPI-1) that are known in the art are used to deliverthe desired gene/nucleic acid.

iPS cells can be generated or derived from terminally differentiatedsomatic cells, as well as from adult stem cells, or somatic stem cells.That is, a non-pluripotent progenitor cell can be rendered pluripotentor multipotent by reprogramming. In such instances, it may not benecessary to include as many reprogramming factors as required toreprogram a terminally differentiated cell. Further, reprogramming canbe induced by the non-viral introduction of reprogramming factors, e.g.,by introducing the proteins themselves, or by introducing nucleic acidsthat encode the reprogramming factors, or by introducing messenger RNAsthat upon translation produce the reprogramming factors (see e.g.,Warren et al., Cell Stem Cell, 2010 Nov. 5; 7(5):618-30, this referenceis incorporated herein by reference in its entirety.). Reprogramming canbe achieved by introducing a combination of nucleic acids encoding stemcell-associated genes including, for example Oct-4 (also known asOct-3/4 or Pouf51), Sox1, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klf1, Klf2,Klf4, Klf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert, and LIN28. In oneembodiment, reprogramming using the methods and compositions describedherein can further comprise introducing one or more of Oct-3/4, a memberof the Sox family, a member of the Klf family, and a member of the Mycfamily to a somatic cell. In one embodiment, the methods andcompositions described herein further comprise introducing one or moreof each of Oct 4, Sox2, Nanog, c-MYC and Klf4 for reprogramming. Asnoted above, the exact method used for reprogramming is not necessarilycritical to the methods and compositions described herein. However,where cells differentiated from the reprogrammed cells are to be usedin, e.g., human therapy, in one embodiment the reprogramming is noteffected by a method that alters the genome. Thus, in such embodiments,reprogramming is achieved, e.g., without the use of viral or plasmidvectors.

The efficiency of reprogramming (i.e., the number of reprogrammed cells)derived from a population of starting cells can be enhanced by theaddition of various small molecules as shown by Shi, Y., et al (2008)Cell-Stem Cell 2:525-528, Huangfu, D., et al (2008) Nature Biotechnology26(7):795-797, and Marson, A., et al (2008) Cell-Stem Cell 3:132-135.This reference is incorporated herein by reference in its entirety.Thus, an agent or combination of agents that enhance the efficiency orrate of induced pluripotent stem cell production can be used in theproduction of patient-specific or disease-specific iPSCs. Somenon-limiting examples of agents that enhance reprogramming efficiencyinclude soluble Wnt, Wnt conditioned media, BIX-01294 (a G9a histonemethyltransferase), PD0325901 (a MEK inhibitor), DNA methyltransferaseinhibitors, histone deacetylase (HDAC) inhibitors, valproic acid,5′-azacytidine, dexamethasone, suberoylanilide hydroxamic acid (SAHA),vitamin C, and trichostatin (TSA), among others.

Other non-limiting examples of reprogramming enhancing agents include:Suberoylanilide Hydroxamic Acid (SAHA (e.g., MK0683, vorinostat) andother hydroxamic acids), BML-210, Depudecin (e.g., (−)-Depudecin), HCToxin, Nullscript(4-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide),Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VPA)and other short chain fatty acids), Scriptaid, Suramin Sodium,Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate,pivaloyloxymethyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin,Depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994(e.g., N-acetyl dinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA(m-carboxycinnaminic acid bishydroxamic acid), JNJ16241199, Tubacin,A-161906, proxamide, oxamflatin, 3-Cl-UCHA (e.g.,6-(3-chlorophenylureido)caproic hydroxamic acid), AOE(2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31 and CHAP 50. Otherreprogramming enhancing agents include, for example, dominant negativeforms of the HDACs (e.g., catalytically inactive forms), siRNAinhibitors of the HDACs, and antibodies that specifically bind to theHDACs. Such inhibitors are available, e.g., from BIOMOL International,Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, AtonPharma, Titan Pharmaceuticals, Schering AG, Pharmion, MethylGene, andSigma Aldrich.

To confirm the induction of pluripotent stem cells for use with themethods described herein, isolated clones can be tested for theexpression of a stem cell marker. Such expression in a cell derived froma somatic cell identifies the cells as induced pluripotent stem cells.Stem cell markers can be selected from the non-limiting group includingSSEA3, SSEA4, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto,Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Nat1. In one embodiment, a cellthat expresses Oct4 or Nanog is identified as pluripotent. Methods fordetecting the expression of such markers can include, for example,RT-PCR and immunological methods that detect the presence of the encodedpolypeptides, such as Western blots or flow cytometric analyses. In someembodiments, detection does not involve only RT-PCR, but also includesdetection of protein markers. Intracellular markers may be bestidentified via RT-PCR, while cell surface markers are readilyidentified, e.g., by immunocytochemistry.

The pluripotent stem cell character of isolated cells can be confirmedby tests evaluating the ability of the iPSCs to differentiate to cellsof each of the three germ layers. As one example, teratoma formation innude mice can be used to evaluate the pluripotent character of theisolated clones. The cells are introduced to nude mice and histologyand/or immunohistochemistry is performed on a tumor arising from thecells. The growth of a tumor comprising cells from all three germlayers, for example, further indicates that the cells are pluripotentstem cells.

Many US patents and patent application Publications teach and describemethods of generating iPSCs and related subject matter. For examples,U.S. Pat. Nos. 9,347,044, 9,347,042, 9,347,045, 9,340,775, 9,341,625,9,340,772, 9,250,230, 9,132,152, 9,045,738, 9,005,975, 9,005,976,8,927,277, 8,993,329, 8,900,871, 8,852,941, 8,802,438, 8,691,574,8,735,150, 8,765,470, 8,058,065, 8,048,675, and US Patent PublicationNos: 20090227032, 20100210014, 20110250692, 20110201110, 20110200568,20110306516, 20100021437, 20110256626, 20110044961, 20120276070,20120263689, 20120128655, 20120100568, 20130295064, 20130029866,20130189786, 20130295579, 20130130387, 20130157365, 20140234973,20140227736, 20140093486, 20140301988, 20140170746, 20140178989,20140349401, 20140065227, and 20150140662. These references areincorporated herein by reference in their entirety.

Somatic Cells for Reprogramming

Somatic cells, as that term is used herein, refer to any cells formingthe body of an organism, excluding germline cells. Every cell type inthe mammalian body—apart from the sperm and ova, the cells from whichthey are made (gametocytes) and undifferentiated stem cells—is adifferentiated somatic cell. For example, internal organs, skin, bones,blood, and connective tissue are all made up of differentiated somaticcells.

Additional somatic cell types for use with the compositions and methodsdescribed herein include: a fibroblast (e.g., a primary fibroblast), amuscle cell (e.g., a myocyte), a cumulus cell, a neural cell, a mammarycell, an hepatocyte and a pancreatic islet cell. In some embodiments,the somatic cell is a primary cell line or is the progeny of a primaryor secondary cell line. In some embodiments, the somatic cell isobtained from a human sample, e.g., a hair follicle, a blood sample, abiopsy (e.g., a skin biopsy or an adipose biopsy), a swab sample (e.g.,an oral swab sample), and is thus a human somatic cell.

Some non-limiting examples of differentiated somatic cells include, butare not limited to, epithelial, endothelial, neuronal, adipose, cardiac,skeletal muscle, skin, immune cells, hepatic, splenic, lung, peripheralcirculating blood cells, gastrointestinal, renal, bone marrow, andpancreatic cells. In some embodiments, a somatic cell can be a primarycell isolated from any somatic tissue including, but not limited tobrain, liver, gut, stomach, intestine, fat, muscle, uterus, skin,spleen, endocrine organ, bone, etc. Further, the somatic cell can befrom any mammalian species, with non-limiting examples including amurine, bovine, simian, porcine, equine, ovine, or human cell. In someembodiments, the somatic cell is a human somatic cell.

When reprogrammed cells are used for generation of thyroid progenitorcells to be used in the therapeutic treatment of disease, it isdesirable, but not required, to use somatic cells isolated from thepatient being treated. For example, somatic cells involved in diseases,and somatic cells participating in therapeutic treatment of diseases andthe like can be used. In some embodiments, a method for selecting thereprogrammed cells from a heterogeneous population comprisingreprogrammed cells and somatic cells they were derived or generated fromcan be performed by any known means. For example, a drug resistance geneor the like, such as a selectable marker gene can be used to isolate thereprogrammed cells using the selectable marker as an index.

Reprogrammed somatic cells as disclosed herein can express any number ofpluripotent cell markers, including: alkaline phosphatase (AP); ABCG2;stage specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60;TRA-1-81; Tra-2-49/6E; ERas/ECAT5, E-cadherin; β-III-tubulin; α-smoothmuscle actin (α-SMA); fibroblast growth factor 4 (Fgf4), Cripto, Dax1;zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Nat1); (ES cellassociated transcript 1 (ECAT1); ESG1/DPPA5/ECAT2; ECAT3; ECAT6; ECAT7;ECAT8; ECAT9; ECAT10; ECAT15-1; ECAT15-2; Fthl17; Sal14;undifferentiated embryonic cell transcription factor (Utf1); Rex1; p53;G3PDH; telomerase, including TERT; silent X chromosome genes; Dnmt3a;Dnmt3b; TRIM28; F-box containing protein 15 (Fbx15); Nanog/ECAT4;Oct3/4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3;CYP25A1; developmental pluripotency-associated 2 (DPPA2); T-celllymphoma breakpoint 1 (Tcl1); DPPA3/Stella; DPPA4; other general markersfor pluripotency, etc. Other markers can include Dnmt3L; Sox15; Stat3;Grb2; β-catenin, and Bmi1. Such cells can also be characterized by thedown-regulation of markers characteristic of the somatic cell from whichthe induced pluripotent stem cell is derived.

Embryonic Bodies (EBs) from Pluripotent Stem Cells

In one embodiment of any one aspect described, the EB are generated orinduced from PSCs, for example, iPSCs or embryonic stem cells (ESCs)derived from the blastocyst stage of embryos from mouse (mESC), primate,and human (hESC) sources.

EBs are three-dimensional aggregates of pluripotent stem cells producedand cultured in vitro in the presence of serum. EBs largely exhibitheterogeneous patterns of differentiated cell types, generating amixture of primitive and definitive hematopoietic progenitor cell types.Primitive progenitors equate to those that arise in vivo naturally inthe earliest stages of embryonic development, whereas at later stages ofmaturation the embryonic populations give rise to definitive progenitorscells, which behave similarly to the cells typical of adulthematopoiesis.

EB formation is often used as a method for initiating spontaneousdifferentiation toward the three germ lineages. EB differentiationbegins with the specification of the exterior cells toward the primitiveendoderm phenotype. The cells at the exterior then deposit extracellularmatrix (ECM), containing collagen IV and laminin, similar to thecomposition and structure of basement membrane. In response to the ECMdeposition, EBs often form a cystic cavity, whereby the cells in contactwith the basement membrane remain viable and those at the interiorundergo apoptosis, resulting in a fluid-filled cavity surrounded bycells. Subsequent differentiation proceeds to form derivatives of thethree germ lineages.

In the absence of supplements, the “default” differentiation of ESCs islargely toward ectoderm, and subsequent neural lineages. However,alternative media compositions, including the use of fetal bovine serumas well as defined growth factor additives, have been developed topromote the differentiation toward mesoderm and endoderm lineages.

As a result of the three-dimensional EB structure, complex morphogenesisoccurs during EB differentiation, including the appearance of bothepithelial- and mesenchymal-like cell populations, as well as theappearance of markers associated with the epithelial-mesenchymaltransition (EMT).

Additionally, the inductive effects resulting from signaling betweencell populations in EBs results in spatially and temporally definedchanges, which promote complex morphogenesis. Tissue-like structures areoften exhibited within EBs, including the appearance of blood islandsreminiscent of early blood vessel structures in the developing embryo,as well as the patterning of neurite extensions (indicative of neuronorganization) and spontaneous contractile activity (indicative ofcardiomyocyte differentiation) when EBs are plated onto adhesivesubstrates such as gelatin. More recently, complex structures, includingoptic cup-like structures were created in vitro resulting from EBdifferentiation.

A non-limited method for producing EBs from iPSC is as follows. HumaniPSCs were differentiated as EBs in the presence of BMP4 and cytokines,as previously described (Chadwick et al., 2003, Blood, 102:906-915).Briefly, iPSC colonies were scraped into non-adherent rotating 10 cmplates. EB media was KO-DMEM+20% FBS (Stem Cell Technologies), 1 mML-glutamine, 1 mM NEAA, penicillin/streptomycin, 0.1 mM3-mercaptoethanol, 200 μg/ml h-transferrin, and 50 μg/ml ascorbic acid.After 24 hrs, media was changed by allowing EBs to settle by gravity,and replaced with EB media supplemented with growth factors: 50 ng/mlBMP4 (R&D Systems), 300 ng/ml SCF, 300 ng/ml FLT3, 50 ng/ml G-CSF, 20ng/ml IL-6, 10 ng/ml IL-3 (all Peprotech). Media was changed on day 3,5, 8 and 10. EBs were observed by day 5. EBs can be selected anddissociated to individual cells anywhere from day 8-14 by digesting withcollagenase B (Roche) for 2 hrs, followed by treatment with enzyme-freedissociation buffer (Gibco), and filtered through an 80 μm filter.

An alternative method of producing EBs from iPSC is as follows.Confluent human or mouse iPSC colonies were disrupted to smallaggregates using collagenase IV (1 mg/ml, 15 minutes at 37° C.). Cellaggregates were resuspended in StemPro fully defined medium (StemPro-34Serum-Free Media, Gibco proprietary formulation, guaranteed as animalprotein-free by the manufacturer) supplemented with 0.5 ng/ml humanrecombinant BMP-4 (R&D Systems) at a ratio of 2 confluent ESC/iPSC wellsfor 1 well of differentiation. During all the differentiation process,cell attachment was prevented using low cluster tissue culture dishes(Corning Costar). On day 1 of differentiation, embryoid bodies (EB) wereharvested and transferred to fresh StemPro media supplemented with 10ng/ml BMP-4 and 5 ng/ml bFGF. After 72 hours, EB were harvested againand transferred to a medium consisting of StemPro media supplementedwith 100 ng/ml human recombinant VEGF (R&D Systems), 5 ng/ml bFGF, 100ng/ml SCF (kind gift from Amgen), 100 ng/ml FLT3-L (kind gift fromAmgen) and 40 ng/ml TPO (Peprotech, UK) for 4 additional days. Alldifferentiation steps were performed in hypoxic conditions (5% O₂) in ahumidified incubator at 37° C. After 8 days of differentiation, EB wereharvested and spun at 65 g for 4 min. Supernatants containingnon-adherent cells were stored on ice and EB were disrupted usingTrypsin-EDTA followed by manual disruption in FBS-containing blockingmedium using a 21G needle. After centrifugation, cells were resuspendedin freshly prepared collagenase IV solution (0.2 mg/ml) for 30 min at37° C. Cells were disrupted again using a 21G needle and filtered over a70 μm cell strainer (Falcon). Supernatants from the initialcentrifugation were pooled with these suspensions and magnetic beadassociated cell sorting (MACS) for the CD34 epitope was performedfollowing manufacturer's instructions (Miltenyi Biotech).

Other methods of generating EBs from ESC and iPSC are known in the art.For example, as described in U.S. Pat. Nos. 6,602,711; 7,220,584;7,452,718; 7,648,833; 7,795,026; 7,803,619; 8,278,097, 8,501,474; and8,986,996, the contents of each are incorporated herein by reference inits entirety.

In one embodiment of any one aspect described, the EB are generated orinduced from PSCs by culturing or exposing the PSCs to morphogens forabout 8 days.

In one embodiment of any one aspect described, the exposure to themorphogens is for about 5-14 days, 5-13 days, 5-12 days, 5-11 days, 5-10days, 5-9 days, 5-8 days, 5-7 days, 5-6 days, 6-14 days, 6-13 days, 6-12days, 6-11 days, 6-10 days, 6-9 days, 6-8 days, 6-7 days, 7-14 days,7-13 days, 7-12 days, 7-11 days, 7-10 days, 7-9 days, 7-8 days, 8-14days, 8-13 days, 8-12 days, 8-11 days, 8-10 days, 8-9 days, 9-14 days,9-13 days, 9-12 days, 9-11 days, 9-10 days, 10-14 days, 10-13 days,10-12 days, 10-11 days, 11-14 days, 11-13 days, 11-12 days, 12-14 days,12-13 days, or 13-14 days.

In one embodiment of any one aspect described, the exposure to themorphogens of the PSCs is for about 5 days, 6 days, 7 days, 9 days, 10days, 11 days 12 days, 13 days or 14 days.

In one embodiment of any one aspect described, the morphogens forinducing EBs formation from PSCs is/are selected from the groupconsisting of holo-transferrin, mono-thioglycerol (MTG), ascorbic acid,bone morphogenetic protein (BMP)-4, basic fibroblast growth factor(bFGF), SB431542, CHIR99021, vascular endothelial growth factor (VEGF),interleukin (IL)-6, insulin-like growth factor (IGF)-1, interleukin(IL)-11, stem cell factor (SCF), erythropoietin (EPO), thrombopoietin(TPO), interleukin (IL)-3, and Fms related tyrosine kinease 3 ligand(Flt-3L).

In some embodiments of any one aspect described, a combination of two ormore of these morphogens are selected to generate EBs from PSCs.

In one embodiment of any one aspect described, a combination of allmorphogens holo-transferrin, MTG, ascorbic acid, BMP-4, bFGF, SB431542,CHIR99021, VEGF, IL-6, IGF-1, IL-11, SCF, EPO, TPO, IL-3, and Flt-3L areused to generate EBs from PSCs.

In one embodiment of any one aspect described, the morphogens forinducing EBs formation from PSCs consist essentially ofholo-transferrin, MTG, ascorbic acid, BMP-4, bFGF, SB431542, CHIR99021,VEGF, IL-6, IGF-1, IL-11, SCF, EPO, TPO, IL-3, and Flt-3L.

In one embodiment of any one aspect described, the morphogens forinducing EBs formation from PSCs consist of holo-transferrin, MTG,ascorbic acid, BMP-4, bFGF, SB431542, CHIR99021, VEGF, IL-6, IGF-1,IL-11, SCF, EPO, TPO, IL-3, and Flt-3L.

In one embodiment of any one aspect described, the method furthercomprises selecting EBs that are generated from the PSCs, prior toisolating HE from the selected EBs.

In one embodiment of any one aspect described, the desired EBs are lessthan 800 microns in size and are selected.

In other embodiments of any one aspect described, the EBs selected areless than 790 μm, less than 780 μm, less than 770 μm, less than 760 μm,less than 750 μm, less than 740 μm, less than 730 μm, less than 720 μm,less than 710 μm, less than 700 μm, less than 690 μm, less than 680 μm,less than 670 μm, less than 660 μm, less than 650 μm, less than 640 μm,less than 630 μm, less than 620 μm, less than 610 μm, less than 600 μm,less than 590 μm, less than 580 μm, less than 570 μm, less than 560 μm,less than 550 μm, less than 540 μm, less than 530 μm, less than 520 μm,less than 510 μm, less than 500 μm, less than 490 μm, less than 480 μm,less than 470 μm, less than 460 μm, less than 450 μm, less than 440 μm,less than 430 μm, less than 420 μm, less than 410 μm, less than 400 μm,less than 390 μm, less than 380 μm, less than 370 μm, less than 360 μm,less than 350 μm, less than 340 μm, less than 330 μm, less than 320 μm,less than 310 μm, less than 300 μm, less than 290 μm, less than 280 μm,less than 270 μm, less than 260 μm, less than 250 μm, less than 240 μm,less than 230 μm, less than 220 μm, less than 210 μm, less than 200 μm,less than 190 μm, less than 180 μm, less than 170 μm, less than 160 μm,less than 150 μm, less than 140 μm, less than 130 μm, less than 120 μm,less than 110 μm, or less than 100 μm in size.

In other embodiments of any one aspect described, the EBs selected areabout 800 μm, about 790 μm, about 780 μm, about 770 μm, about 760 μm,about 750 μm, about 740 μm, about 730 μm, about 720 μm, about 710 μm,about 700 μm, about 690 μm, about 680 μm, about 670 μm, about 660 μm,about 650 μm, about 640 μm, about 630 μm, about 620 μm, about 610 μm,about 600 μm, about 590 μm, about 580 μm, about 570 μm, about 560 μm,about 550 μm, about 540 μm, about 530 μm, about 520 μm, about 510 μm,about 500 μm, about 490 μm, about 480 μm, about 470 μm, about 460 μm,about 450 μm, about 440 μm, about 430 μm, about 420 μm, about 410 μm,about 400 μm, about 390 μm, about 380 μm, about 370 μm, about 360 μm,about 350 μm, about 340 μm, about 330 μm, about 320 μm, about 310 μm,about 300 μm, about 290 μm, about 280 μm, about 270 μm, about 260 μm,about 250 μm, about 240 μm, about 230 μm, about 220 μm, about 210 μm,about 200 μm, about 190 μm, about 180 μm, about 170 μm, about 160 μm,about 150 μm, about 140 μm, about 130 μm, about 120 μm, about 110 μm, orabout 100 μm in size.

The EB size selection can be achieved by any method known in the art.For example, by filtration through a filter with the desired pore size,e.g., a 200 μm filter. For example, selection is done visually.

In one embodiment of any one aspect described, the EB cells within theEBs selected are compactly adhered to each other and are dissociated toindividual EB cells.

In one embodiment of any one aspect described, the EB cells within theEBs selected are dissociated to individual EB cells by digestion withtrypsin or collagenase.

In one embodiment of any one aspect described, the EB cells of theselected EBs are dissociated to individual EB cells prior to theisolation of HE.

Hemogenic Endothelia Cells (HE) Isolated from the Dissociated IndividualEB Cells

Hemogenic endothelium (HE) is a special subset of endothelial cellsscattered within blood vessels that can differentiate into hematopoieticcells. During embryonic development, multilineage HSCs/progenitor cellsare derived from specialized endothelial cells, termed hemogenicendothelium, within the yolk sac, placenta, and aorta. The multilineageHSCs/progenitor cells responsible for the generation of all blood celltypes during definitive hematopoiesis arise from hemogenic endothelium.All blood cells emerge from hemogenic endothelial-expressing cellsthrough an endothelial-to-hematopoietic transition (EHT).

In vitro differentiation EB produces a heterogeneous population ofcells, including cells that are hemogenic endothelial-like. These arethe HE cells that are isolated and induced to under go EHT in culture.In one embodiment of any one aspect described, the HE are isolated fromthe selected and dissociated EB cells.

In one embodiment of any one aspect described, the HE are isolatedimmediately from the selected and dissociated EB cells. In otherembodiments of any one aspect described, the HEs are isolated less thanthree hours, less than two hours, less than one hour, less than 55 min,less than 50 min, less than 45 min, less than 40 min, less than 35 min,less than 30 min, less than 25 min, less than 20 min, less than 15 min,and less than 10 min after the dissociation of the select EBs toindividual EB cells. In other embodiments of any one aspect described,the HEs are isolated within three hours, within two hours, within onehour, within 55 min, within 50 min, within 45 min, within 40 min, within35 min, within 30 min, within 25 min, within 20 min, within 15 min, andwithin 10 min after the dissociation of the select EBs to individual EBcells.

In one embodiment of any one aspect described, the isolated HE aredefinitive HE. Definitive HE is a population that is defined bycombination of surface antigen markers CD34+FLK+CD235A−CD43−.

Definitive hematopoietic stem cells (HSCs) are the cells responsible forthe continuous production of all mature blood cells during the entireadult life span of an individual. Similarly, definitive HE are the cellsthat are responsible for the continuous production of all mature bloodcells during the entire adult life span of an individual.

In one embodiment of any one aspect described, the isolated HE areFLK1+, CD34+, CD43− and CD235A−.

In one embodiment of any one aspect described, the isolated HE areFlk1+cKit+CD45−.

These are the cell surface biomarkers on the isolated HE before theendothelial-to-hematopoietic transition (EHT).

The HEs can be isolated by any method known in the art. For example, byimmune-magnetic beads directed to the cell surface biomarkers that arecharacteristics of HEs. For example, by positive selection for FLK1+ andCD34+ cells from the dissociated individual EB cells followed bynegative selection for CD43+ cells, and for CD235A+ cells from theresultant FLK1+ and CD34+ cells. FLK1+ and CD34+ cells that are CD43+are discarded. FLK1+ and CD34+ cells that are CD235A+ are alsodiscarded. The remaining FLK1+ and CD34+ cells are therefore CD43− andCD235A− cells, the desired HEs.

Other methods of generating HEs from ESC and iPSC are known in the art.For example, as described in U.S. Pat. No. 9,382,531, the contents ofwhich is incorporated herein by reference in its entirety.

Endothelial-to-Hematopoietic Transition (EHT)

The endothelial to hematopoietic transition (EHT) is a key developmentalevent leading to the formation of blood stem and progenitor cells duringembryogenesis. In embryogenesis, the development of hematopoietic cellsin the embryo proceeds sequentially from mesoderm through thehemangioblast to the hemogenic endothelium (HE) and hematopoieticprogenitors cells. The HE then undergoes the EHT by becomingpre-hematopoietic stem and progenitor cells (Pre-HSPC). Eventually afterlosing all their endothelial characteristics they become HSPC.

In vitro culture, it is possible to induce EHT in HE with certainfactors and cytokines. Combination of all these factors is required forefficient achievement of EHT.

In one embodiment of any one aspect described, the EHT occurs byculturing or contacting the isolated HE in culture with thrombopoietin(TPO), interleukin (IL)-3, stem cell factor (SCF), IL-6, IL-11,insulin-like growth factor (IGF)-1, erythropoietin (EPO), vascularendothelial growth factor (VEGF), basic fibroblast growth factor (bFGF),bone morphogenetic protein (BMP)4, Fins related tyrosine kinase 3 ligand(Flt-3L), sonic hedgehog (SHH), angiotensin II, and chemical AGTR1(angiotensin II receptor type I) blocker losartan potassium.

In one embodiment of any one aspect described, the isolated HEs areincubated in the EHT media for a period of time.

In one embodiment of any one aspect described, the incubation of theisolated HE in the EHT media is for about 3-10 days.

In one embodiment of any one aspect described, the incubation of theisolated HE in the EHT media is for about 3-9 days, 3-8 days, 3-7 days,3-6 days, 3-5 days, 3-4 days, 4-10 days, 4-9 days, 4-8 days, 4-7 days,4-6 days, 4-5 days, 5-10 days, 5-9 days, 5-8 days, 5-7 days, 5-8 days,5-7 days, 5-6 days, 6-10 days, 6-9 days, 6-8 days, 6-7 days, 7-10 days,7-9 days, 7-8 days, 8-10 days, 8-9 days, or 9-10 days.

In one embodiment of any one aspect described, incubation of theisolated HE in the EHT media is for about 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, or 10 days.

To determine that EHT has occurred in the HE, morphological detection ofround cells under microscopy was performed. In addition, FACS analysisof surface markers CD34+CD45+ is performed.

In one embodiment of any one aspect described, the isolated HE that haveundergone EHT exhibit round cell morphology.

Transcription Factors that Induced Differentiation of HE toMulti-Lineage HSCs and HSPCs

To specifying HSCs and HSPCs from the HE having undergone EHT, theinventors use a strategy that is defined as “respecification”.Respecification combines directed differentiation withtranscription-based reprogramming to re-establish HSC fate. Themolecular differences between primary human HSCs and progenitors havebeen well characterized by gene expression profiling, providing arational approach to introduce stem cell genes back into progenitors.The inventors were able to obtain transplantable HSC by restoring theHSC transcription factor network in the HE derived from hPSCs.

The inventors tailored transcription factor combinations for the HE. Theminimally required five transcription factors: ERG, HOXA9, HOXA5, LCORand RUNX1. Additionally, HOXA10 and SPI1 transcription factors can beused to induce differentiation of the HE to multilineage hematopoieticprogenitors.

Generation of iPSCs by somatic cell reprogramming involves globalepigenetic remodeling, and chromatin-modifying enzymes have beencharacterized as barriers or facilitators of reprogramming. Within thehematopoietic system, there are many epigenetic changes that mediateblood development during ontogeny and differentiation from HSCs tomature progeny. The progression from HSCs to differentiated progenyinvolves coordinated control of gene expression programs leading to theactivation or repression of lineage-specific genes. The events that leadto the formation of all mature hematopoietic cells involve regulation ofboth gene expression and DNA recombination, mainly through the controlof chromatin accessibility. HSC state is controlled by a large number oftranscription factors and epigenetic modifiers. The inventors usedscreening strategies find additional factors that regulate of the HSCfate.

Accordingly, in one embodiment of any method, cells, or compositiondescribed herein, the multilineage hematopoietic progenitor cells aregenerated by introducing in vitro an exogenous gene coding copy each ofthe following transcription factors: ERG, HOXA9, HOXA5, LCOR and RUNX1,into the HE, the HE having undergone EHT and exhibit round cellmorphology. In one embodiment, a vector is used as the transport vehicleto introduce any of the herein described exogenous gene coding copiesinto the HE. For example, by transfecting the HE with a vector or more,wherein the vector(s) collectively carry an exogenous gene coding copyof each of the following transcription factors, ERG, HOXA9, HOXA5, LCORand RUNX1, for the in vivo expression of the transcription factor in thetransfected cells. For example, by contacting the HE with a vector ormore, wherein the vector(s) collectively carry an exogenous gene codingcopy of each of the following transcription factors, ERG, HOXA9, HOXA5,LCOR and RUNX1, for the in vivo expression of the transcription factorin the contacted cells. For example, by contacting the isolated HE witha nucleic acid or more, wherein the nucleic acid (s) collectively carryan exogenous gene coding copy of each of the following transcriptionfactors, ERG, HOXA9, HOXA5, LCOR and RUNX1, for the in vivo expressionof the transcription factor in the contacted cells.

In one embodiment of any method, cells, or composition described herein,the multilineage hematopoietic progenitor cells are generated bycontacting a population of HE with a vector or more, wherein thevector(s) collectively carrying an exogenous gene coding copy of each ofthe following transcription factors, ERG, HOXA9, HOXA5, LCOR and RUNX1,for the in vivo expression of the factors in the contacted cells, andwherein the transfected transcription factors are expressed in vivo inthe contacted cells. The contacting is in vitro or ex vivo.

In one embodiment of any method, cells, or composition described herein,the multilineage hematopoietic progenitor cells are generated bycontacting the HE with a nucleic acid or more, wherein the nucleic acid(s) collectively comprises an exogenous gene coding copy of each of thefollowing transcription factors, ERG, HOXA9, HOXA5, LCOR and RUNX1, forthe in vivo expression of the transcription factor in the contactedcells. The contacting is in vitro or ex vivo.

In one embodiment of any method, cells, or composition described herein,the contacting of the HE with any vector(s), nucleic acid(s) orcompositions comprising the vector(s) or nucleic acid(s) describedherein occurs in vitro or ex vivo.

In one embodiment of any methods, cells, or composition describedherein, the contacting or introduction is repeated at least once. In oneembodiment of any methods, cells, or composition described herein, thecontacting or introduction is repeated twice or more times.

In one embodiment of any method, cells, or composition described herein,the method further comprising transfecting the HE with an exogenous genecoding copy of the transcription factor, HOXA10 or SPI1 or both HOXA10and SPI1, wherein the transfected transcription factor(s) is/areexpressed in vivo in the transfected cells. The transfecting is in vitroor ex vivo.

In one embodiment of any method, cells, or composition described herein,the method further comprising transfecting the HE with an exogenous genecoding copy of the transcription factors, HOXA10, wherein thetransfected transcription factor is expressed in vivo in the transfectedcells.

In one embodiment of any method, cells, or composition described herein,the method further comprising transfecting the HE with an exogenous genecoding copy of the transcription factors, SPI1, wherein the transfectedtranscription factor is expressed in vivo in the transfected cells.

In one embodiment of any one aspect described, the disclosedtranscription factors are expressed in the transfected cells.

In another embodiment of any one aspect described, the disclosedtranscription factors are expressed in the engineered cells.

In one embodiment of any one aspect described, the expression of thedisclosed transcription factors in the transfected or engineered cellsproduces a population of multi-lineage HSCs and HSPCs.

Transcription Factors

Runt Related Transcription Factor 1 (RUNX1) is the alpha subunit 2 of aheterodimeric transcription factor that binds to the core element ofmany enhancers and promoters. The protein encoded by this gene RUNX1represents the alpha subunit of the heterodimeric transcription factorand is thought to be involved in the development of normalhematopoiesis. Chromosomal translocations involving this gene arewell-documented and have been associated with several types of leukemia.Three transcript variants encoding different isoforms have been foundfor this gene. RUNX1 is essential for hematopoietic commitment of HE andcan convert endothelial cells to hematopoietic progenitor cells.

The REFSEQ mRNAs for RUNX1 are NM_001001890.2; NM_001122607.1;NM_001754.4; XM_005261068.3; ani XM_005261069.4.

Ligand Dependent Nuclear Receptor Corepressor (LCOR) is atranscriptional corepressor widely expressed in fetal and adult tissuesthat is recruited to agonist-bound nuclear receptors through a singleLxxLL motif, also referred to as a nuclear receptor (NR) box. LCOR is acomponent of histone deacetylation complex, is mutated in B-celllymphoma, indicating a role in B-lymphopoiesis, but this factor has notpreviously been implicated in HSC functions and its role remains to bedefined. LCOR may act as transcription activator that binds DNA elementswith the sequence 5-CCCTATCGATCGATCTCTACCT-3 (By similarity). It is arepressor of ligand-dependent transcription activation by target nuclearreceptors, repressing of ligand-dependent transcription activation byESR1, ESR2, NR3C1, PGR, RARA, RARB, RARG, RXRA and VDR. The REFSEQ mRNAsfor LCOR are NM_015652.3; NM_001170765.1; NM_001170766.1;NM_001346516.1; and NM_032440.3.

ERG (ETS-related gene) is an oncogene meaning that it encodes a proteinthat typically is mutated in cancer. ERG is a member of the ETS(erythroblast transformation-specific) family of transcription factors.The ERG gene encodes for a protein, also called ERG that functions as atranscriptional regulator. Genes in the ETS family regulate embryonicdevelopment, cell proliferation, differentiation, angiogenesis,inflammation, and apoptosis. The external idenifications for ERG geneare as follows: HGNC: 3446; Entrez Gene: 2078; Ensembl: ENSG00000157554;OMIM: 165080; UniProtKB: P11308; EMBL: AY204741 mRNA and thecorresponding mRNA translation: AAP41719.1; and GENBANK: AY204742 mRNAand the corresponding mRNA translation: AAP41720.1.

Spi-1 Proto-Oncogene (SPI1, also known as PU.1) is required forhematopoietic progenitor cell emergence and regulates myeloidspecification. The oncogene is an ETS-domain transcription factor thatactivates gene expression during myeloid and B-lymphoid celldevelopment. The nuclear protein binds to a purine-rich sequence knownas the PU-box found near the promoters of target genes, and regulatestheir expression in coordination with other transcription factors andcofactors. The protein can also regulate alternative splicing of targetgenes. Multiple transcript variants encoding different isoforms havebeen found for this gene. The external idenifications for SPI1 gene isas follows: HGNC: 11241; Entrez Gene: 6688; Ensembl: ENSG00000066336;OMIM: 165170; and UniProtKB: P17947. The REFSEQ mRNAs for SPI1 areNM_001080547.1; NM_003120.2; XM_011520307.1 and XM_017018173.1.

Homeobox protein Hox-A9 is a protein that in humans is encoded by theHOXA9 gene. In vertebrates, the genes encoding the homeobox genes classof transcription factors are found in clusters named A, B, C, and D onfour separate chromosomes. Expression of these proteins is spatially andtemporally regulated during embryonic development. Hox-A9 is part of theA cluster on chromosome 7 and encodes a DNA-binding transcription factorwhich may regulate gene expression, morphogenesis, and differentiation.The external idenifications for HOXA9 gene are as follows: HGNC: 5109;Entrez Gene: 3205; Ensembl: ENSG00000078399; OMIM: 142956; UniProtKB:P31269; EMBL: BT006990 mRNA and the corresponding mRNA translation:AAP35636.1; and GENBANK:AC004080 Genomic DNA. The REFSEQ mRNAs for HOXA9is NM_152739.3

HOX family members have been reproducibly implicated in hematopoiesisacross species. HOXA9 is the key homeotic gene that defines HSCidentity, interacting with ERG to support HSC renewal duringembryogenesis and stress hematopoiesis, indicating a basis for thefunctional cooperation of HOXA9 and ERG in the system presented herein.HOXA5 is a transcriptional target of Notch signaling in T-cellprogenitors along with HOXA9 and HOXA10, consistent with a role inT-lymphopoiesis. These factors share binding sites in the genome andcooperate to recruit chromatin modulators (e.g. RUNX1 and HOXA families)to induce and maintain HSPCs. The external idenifications for HOXA5 geneare as follows: HGNC: 5106; Entrez Gene: 3202; Ensembl: ENSG00000106004;OMIM: 142952; and UniProtKB: P20719. The REFSEQ mRNAs for HOXA5 isNM_019102.3. The external idenifications for HOXA5 gene are as follows:HGNC: 5100; Entrez Gene: 3206; Ensembl: ENSG00000253293; OMIM: 142957,and UniProtKB: P31260. The REFSEQ mRNAs for HOXA10 are NM_018951.3 andNM_153715.3.

HOX- and ETS-family transcription factors HOXA9 and ERG are inducers ofself-renewal and multilineage potential in hematopoietic progenitorsdifferentiated from hPSCs. RORA is a nuclear receptor that plays a rolein maintaining quiescence of hematopoietic progenitors. The addition ofSOX4 and MYB modulates this network to enable myeloid and erythroidengraftment in vivo.

OCT4, SOX2, KLF4 and c-MYC are the original four transcription factorsidentified to reprogram mouse fibroblasts into iPSCs. These same fourfactors were also sufficient to generate human iPSCs. OCT3/4 and SOX2function as core transcription factors of the pluripotency network byregulating the expression of pluripotency-associated genes. Krüppel-likefactor 4 (KLF4) is a downstream target of LIF-STAT3 signaling in mouseES cells and regulates self-renewal. Human iPSCs can also be generatedusing four alternative factors; OCT4 and SOX2 are required but KLF4 andc-MYC could be replaced with NANOG, a homeobox protein important for themaintenance of pluripotency in both ES cells and early embryos, andLIN28, an RNA binding protein.

Transcription factor SOX-4 (SOX4). This intronless gene encodes a memberof the SOX (SRY-related HMG-box) family of transcription factorsinvolved in the regulation of embryonic development and in thedetermination of the cell fate. The encoded protein act as atranscriptional regulator after forming a protein complex with otherproteins, such as syndecan binding protein (syntenin). The protein mayfunction in the apoptosis pathway leading to cell death as well as totumorigenesis and may mediate downstream effects of parathyroid hormone(PTH) and PTH-related protein (PTHrP) in bone development. The externalidenifications for Homo sapiens (Human) SOX4 gene are as follows: HGNC:11200; Entrez Gene: 6659; Ensembl: ENSG00000124766; OMIM: 184430;UniProtKB: Q06945; EMBL: BC072668 mRNA mRNA and the corresponding mRNAtranslation: AAH72668.1; GENBANK: X65661 mRNA and the corresponding mRNAtranslation: CAA46612.1.

The cDNA encoding the described and desired transcription factors can becloned by methods known in the art into expression vectors for in vivoexpression in the cells. The expression vectors can be constitutive orinducible vectors. The protein and DNA information for transcriptionfactors can be found in the publically available databases such as theGenBank™ database on the National Institute of Health, the UniProt atthe Protein knowledgebase, and GeneCard database at the WeizmannInstitute for Science. The cDNA clones or plasmids carrying the cDNA canbe purchased at BioCat GmbH, and the lentivirus carrying the cDNAs forexpression can also be purchased at Applied Biological Materials (ABM)Inc.

In one embodiment of any method, cells, or composition described herein,a vector is used as a transport vehicle to introduce any of the hereindescribed exogenous gene coding copies of transcription factors orreprogramming factors or nucleic acid inhibitor into the target cellsselected from the disclosed HE.

In one embodiment of any method, cells, or composition described herein,a vector is used as a transport vehicle to introduce any of the hereindescribed nucleic acid comprising the described exogenous gene codingcopies of transcription factors or reprogramming factors or nucleic acidinhibitor into the target cells selected from the disclosed HE.

In one aspect, the present specification provides a vector or more,wherein the vector(s) collectively comprises an exogenous gene codingcopies of each of the transcription factors or reprogramming factors ornucleic acid inhibitor described. The exogenous gene coding copy is forthe expression of transcription factors or reprogramming factors insidethe cells. In one embodiment, each vector consists essentially of atranscription factors or reprogramming factor described herein. In oneembodiment, each vector consists essentially of two or more of thedescribed transcription factors or reprogramming factors.

In one aspect, the present specification provides a vector or more,wherein the vector(s) collectively comprises nucleic acids comprisingthe described exogenous gene coding copies of transcription factors orreprogramming factors or nucleic acid inhibitor. The nucleic acid is forthe expression of the transcription factors or reprogramming factorsinside the cells.

In one aspect, the present specification provides a vector or more,wherein the vector(s) collectively comprises an exogenous gene codingcopy of each of the following transcription factors, ERG, HOXA9, HOXA5,LCOR and RUNX1 described herein. In another aspect, the vector(s)collectively further comprise an exogenous gene coding copy of HOXA10and SPI1.

In another aspect, the disclosed herein also provides a host cellcomprising a vector or more described herein or nucleic acid(s) of thetranscription factors or reprogramming factors or both described herein.

In another aspect, the disclose also provides a host cell comprising avector or more described herein or nucleic acid(s) of the transcriptionfactors, ERG, HOXA9, HOXA5, LCOR and RUNX1 described herein.

In another aspect, the host cell further comprises a vector or moredescribed herein or nucleic acid(s) of the transcription factors HOXA10and SPI1.

In another aspect, the host cell further comprises a vector or moredescribed herein or nucleic acid(s) of reprogramming factors or bothdescribed herein, OCT4, SOX2, and KLF4, and optionally with c-MYC ornanog and LIN28. For example, the one or more vectors collectively carrythe nucleic acids of the reprogramming factors OCT4, SOX2, NANOG, andLIN28, or collectively carry the nucleic acids of the reprogrammingfactors OCT4, SOX2, and KLF4, or OCT4, SOX2, KLF4, and c-MYC.

In one embodiment of any method, cells, or composition described herein,the vector further comprises a spleen focus-forming virus promoter, atetracycline-inducible promoter, a Doxycycline (Dox)-inducible, or aβ-globin locus control region and a β-globin promoter. In oneembodiment, the promoter provided for targeted expression of the nucleicacid molecule therein. Other examples of promoters include but are notlimited to the CMV promoter and EF1α promoters for the varioustransgenes.

In one embodiment of any method, cells, or composition described herein,the vector is a virus.

In one embodiment of any method, cells, or composition described herein,the vector is an episomal vector.

In one embodiment of any method, cells, or composition described herein,the vector is a non-integrative episomal vector.

Non-limiting examples of non-integrative episomal vectors known in theart are oriP/EBNA-1 [Epstein Barr nuclear antigen-1], and the non-viralepisomal vector pEPI-1, and those disclosed herein, and in the U.S. Pat.Nos. 5,674,703, 5,624,820, 5,830,725, 5,976,807, 6,077,992, 6,110,490,6,255,071, 6,436,392, 6,635,472, 6,642,051, 6,632,980, 6,808,923, and8,187,836, and in the US patent publication numbers: US20020123034,US20100184227, US20020119570, and US20040161741, the contents are herebyincorporated by reference in their entirety.

Episomal vectors for use in, e.g., gene therapy, is further reviewed in,e.g., Ehrhardt, A., et al. Current Gene Therapy. 2008; 8: 147-161, whichis incorporated herein by reference. Episomal vectors for use in, e.g.,gene expression, is further reviewed in, e.g., Van Craenenbroeck, K., etal. Eur. J. Biochem. 2000; 267: 5665-5678, which is incorporated hereinby reference.

Episomal iPSC reprogramming vectors are commercially available, e.g.,via ThermoFisher Scientific (Waltham, A). Information regarding theseepisoma; iPSC reprogamming vectors can be found on the world wide wibe,e.g., at www.thermofisher.com/order/catalog/product/A14703.

A DNA molecule that replicates independently of chromosomal DNA is anepisome. By this definition a plasmid is (usually) an episome. If aplasmid integrates into a chromosome by some mechanism (as for examplein Hfr strains of E. coli where the F plasmid is integrated) the plasmidloses its episomal status.

In one embodiment of any method, cells, or composition described herein,the episomal vector described herein for the exogenous gene transferdoes not integrate into the chromosomal DNA of the host cells, e.g., theiPSCs, or EBs or hemogenic endothelia cells (HEs) or HEs that haveundergone EHT.

In one embodiment of any method, cells, or composition described herein,the episomal vector described herein for the exogenous gene transfercontains a mammalian origin of replication.

In one embodiment of any method, cells, or composition described herein,the vector is a non-integrative oriP/EBNA-1 based vector, a non-viralepisomal vector pEPI-1, or a replication deficient AAV, or a S/MAR-basedvector or mammalian/human artificial chromosomes (MAC/HAC) describedherein.

A episomal vector can encode a single transcription vector requested inthe invention described herein (e.g., ERG, HOXA9, HOXA5, LCOR or RUNX1).Alternatively, a episomal vector can encode at least 2 or moretranscription factors. In one embodiment, the non-integrative vector(e.g., an episomal vector) encodes one transcription factor. In anotherembodiment, the non-integrative vector (e.g., an episomal vector)encodes at least two, at least three, at least four, at least five ormore transcription factors of the invention described herein (e.g., ERG,HOXA9, HOXA5, LCOR or RUNX1).

In one embodiment of any method, cells, or composition described herein,the virus is a lentivirus, an adenovirus, an adeno-associated virus, apox virus, an alphavirus, or a herpes virus. In one embodiment of anymethod, cells, or composition described herein, the virus is an avianviral vector.

In one embodiment of any method, cells, or composition described herein,the in vivo expression of the described transcription factors areregulatable. That is, the promoters used in the vectors for geneexpression are inducible.

In one aspect of any method, cells, or composition described herein, thelentivirus is selected from the group consisting of: humanimmunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type2 (HIV-2), caprine arthritis-encephalitis virus (CAEV), equineinfectious anemia virus (EIAV), feline immunodeficiency virus (FIV),bovine immune deficiency virus (BIV), and simian immunodeficiency virus(SIV).

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional nucleic acid segments canbe ligated. Another type of vector is a viral vector, wherein additionalnucleic acid segments can be ligated into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors having a bacterial originof replication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “recombinant expression vectors”,or more simply “expression vectors.” In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” can be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the methods and compositions described herein can include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, lentiviruses, adenoviruses andadeno-associated viruses), which serve equivalent functions.

Within an expression vector, “operably linked” is intended to mean thatthe nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner which allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in atarget cell when the vector is introduced into the target cell). Theterm “regulatory sequence” is intended to include promoters, enhancersand other expression control elements (e.g., polyadenylation signals).Such regulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcell and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences).Furthermore, the DNA-targeting endonuclease can be delivered by way of avector comprising a regulatory sequence to direct synthesis of theDNA-targeting endonuclease at specific intervals, or over a specifictime period. It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the target cell, the level of expression desired, and the like.

Suitable viral vectors include, but are not limited to, vectors based onRNA viruses, such as retrovirus-derived vectors (for example, Moloneymurine leukemia virus (MLV)-derived vectors), and more complexretrovirus-derived vectors (such as Lentivirus-derived vectors); andvectors based on DNA viruses, such as adenovirus-based vectors andadeno-associated virus (AAV)-based vectors. In some embodiments, thepolynucleotide delivery system comprises a retroviral vector, morepreferably a lentiviral vector. Non-limiting examples of viral vectorinclude lentivirus vectors derived from human immunodeficiency virus 1(HIV-1), HIV-2, feline immunodeficiency virus (FIV), equine infectiousanemia virus, simian immunodeficiency virus (SIV) and maedi/visna virus.

In one embodiment of any one aspect described, the population ofmulti-lineage HSCs and HSPCs, produced by the expression of thedisclosed transcription factors in the transfected or engineered cells,engrafts in vivo in the recipient subject and produces blood cells invivo.

In one embodiment of any one aspect described, the population ofmulti-lineage HSCs and HSPCs, produced by the expression of thedisclosed transcription factors in the transfected or engineered cells,reconstitutes the hematopoietic system in vivo in the recipient subject.

In one embodiment of any one aspect described, the engineered cellcomprises an exogenous copy of each of the following transcriptionfactors ERG, HOXA9, HOXA5, LCOR and RUNX1.

In one embodiment of any one aspect described, the engineered cellfurther comprises an exogenous copy of each of the followingreprogramming factors OCT4, SOX2, KLF4 and optionally c-MYC.

In one embodiment of any one aspect described, the engineered cellfurther comprises an exogenous copy of each of the followingreprogramming factors OCT 4, SOX2, NANOG, and LIN28.

In one embodiment of any one aspect described, the composition ofengineered cells further comprises a pharmaceutically acceptablecarrier.

In one embodiment of any one aspect described, the subject is patientswho has undergone chemotherapy or irradiation or both, and manifestdeficiencies in immune or blood function or lymphocyte reconstitution orboth deficiencies in immune function and lymphocyte reconstitution.

In one embodiment of any one aspect described, the subject prior toimplantation, the immune cells are treated ex vivo with prostaglandin E2and/or antioxidant N-acetyl-L-cysteine (NAC) to promote subsequentengraftment in a recipient subject.

In one embodiment of any one aspect described, the disclosed engineeredcells are autologous to the recipient subject or at least HLA typematched with the recipient subject.

It is also envisioned that the methods described herein can be used asprophylaxis

Uses of Engineered Immune Cells Derived from Pluripotent Stem Cells

The engineered cells described herein are useful in the laboratory forbiological studies. For examples, these cells can be derived from anindividual having a genetic disease or defect, and used in thelaboratory to study the biological aspects of the diseases or defect,and to screen and test for potential remedy for that diseases or defect.Contemplated but not limited to are congenital hematologic disorderssuch as Diamond-Blackfan-Anemia, Shwatchman-Diamond-Anemia, and immunedeficiency such as Omenn syndrome.

Alternatively, the engineered cells described herein are useful incellular replacement therapy in subjects having the need. For example,patients who have undergone chemotherapy. For example, patients who haveundergone chemotherapy or irradiation or both, and manifest deficienciesin immune function and/or lymphocyte reconstitution. For example,cellular replacement therapy can be performed in subjects withcongenital hematologic disorders described herein.

In various embodiments, the engineered cells described herein areadministered (ie. implanted or transplanted) to a subject in need ofcellular replacement therapy. The implanted engineered cell engrafts andreconstitutes the hematopoietic system in the recipient subject.

As used herein, the terms “administering,” “introducing” and“transplanting” are used interchangeably in the context of the placementof described cells, e.g. HSC, HSPC, hematopoietic progenitor cells, intoa subject, by a method or route which results in at least partiallocalization of the introduced cells at a desired site, such as a siteof injury or repair, such that a desired effect(s) is produced. Thecells e.g. HSC, HSPC, hematopoietic progenitor cells, or theirdifferentiated progeny can be administered by any appropriate routewhich results in delivery to a desired location in the subject where atleast a portion of the implanted cells or components of the cells remainviable.

In various embodiments, the engineered cells described herein areoptionally expanded ex vivo prior to administration to a subject. Inother embodiments, the engineered cells are optionally cryopreserved fora period, then thawed prior to administration to a subject.

The engineered cells used for cellular replacement therapy can beautologous/autogeneic (“self”) or non-autologous (“non-self,” e.g.,allogeneic, syngeneic or xenogeneic) in relation to the recipient of thecells. “Autologous,” as used herein, refers to cells from the samesubject. “Allogeneic,” as used herein, refers to cells of the samespecies that differ genetically to the cell in comparison. “Syngeneic,”as used herein, refers to cells of a different subject that aregenetically identical to the cell in comparison. “Xenogeneic,” as usedherein, refers to cells of a different species to the cell incomparison. In other embodiments, the engineered cells of theembodiments of the present disclosure are allogeneic.

In various embodiments, the engineered cell described herein that is tobe implanted into a subject in need thereof is autologous or allogeneicto the subject.

In various embodiments, the engineered cell described herein can bederived from one or more donors, or can be obtained from an autologoussource. In some embodiments of the aspects described herein, theengineered cells are expanded in culture prior to administration to asubject in need thereof.

In various embodiments, the engineered cell described herein can bederived from one or more donors, or can be obtained from an autologoussource.

In various embodiments, prior to implantation, the recipient subject istreated, or was previously been treated with chemotherapy and/orradiation.

In one embodiment, the chemotherapy and/or radiation are to reduceendogenous stem cells in the recipient subject to facilitate engraftmentof the implanted cells.

In one embodiment, the chemotherapy and/or radiation have reduced theability of the recipient subject to synthesize sufficient hematopoieticcells to sustain life, e.g., inability to make blood cells, plateletcells, etc.

In various embodiments, prior to implantation, the engineered immunecells or the inhibited, reverse-lineage multilineage hematopoieticprogenitor cells are treated ex vivo with prostaglandin E2 and/orantioxidant N-acetyl-L-cysteine (NAC) to promote subsequent engraftmentin a recipient subject.

In various embodiments, the recipient subject is a human.

In various embodiments, the subject is diagnosed with HIV, ahematological disease, undergoing a cancer treatment, or an autoimmunedisease. For example, the subject has aplastic anemia, X-linkedlymphopenic immune deficiency, Wiskott-Aldrich syndrome, Fanconi anemia,cancer, or acute lymphoblastic leukemia (ALL).

In one aspect of any method, cells and composition described herein, asubject is selected to donate a somatic cell which would be used toproduce iPSCs and an engineered immune cell described herein. In oneembodiment, the selected subject has a genetic disease or defect.

In various embodiments, the donor subject is a human.

In various embodiments, the donor or the recipient subject is an animal,human or non-human, and rodent or non-rodent. For example, the subjectcan be any mammal, e.g., a human, other primate, pig, rodent such asmouse or rat, rabbit, guinea pig, hamster, cow, horse, cat, dog, sheepor goat, or a non-mammal such as a bird.

In various embodiments, the donor or the recipient subject is diagnosedwith HIV, a hematological disease or cancer.

In one aspect of any method, cells and composition described herein, abiological sample or a population of embryonic stem cells, somatic stemcells, progenitor cells, bone marrow cells, hematopoietic stem cells, orhematopoietic progenitor cells is obtained from the donor subject.

In various embodiments, biological sample or a population of embryonicstem cells, somatic stem cells, progenitor cells, bone marrow cells,hematopoietic stem cells, or hematopoietic progenitor cells describedherein can be derived from one or more donors, or can be obtained froman autologous source.

In one embodiment, the embryonic stem cells, somatic stem cells,progenitor cells, bone marrow cells, hematopoietic stem cells,hematopoietic progenitor cells are isolated from the donor subject,transfected, cultured (optional), and transplanted back into the samesubject, i.e. an autologous cell transplant. Here, the donor and therecipient subject is the same individual. In another embodiment, theembryonic stem cells, somatic stem cells, progenitor cells, bone marrowcells, hematopoietic stem cells, or hematopoietic progenitor cells areisolated from a donor who is an HLA-type match with a subject(recipient). Donor-recipient antigen type-matching is well known in theart. The HLA-types include HLA-A, HLA-B, HLA-C, and HLA-D. Theserepresent the minimum number of cell surface antigen matching requiredfor transplantation. That is the transfected cells are transplanted intoa different subject, i.e., allogeneic to the recipient host subject. Thedonor's or subject's embryonic stem cells, somatic stem cells,progenitor cells, bone marrow cells, hematopoietic stem cells, orhematopoietic progenitor cells can be transfected with a vector ornucleic acid comprising the nucleic acid molecule(s) described herein,the transfected cells are cultured, inhibited, and differentiated asdisclosed, optionally expanded, and then transplanted into the recipientsubject. In one embodiment, the transplanted engineered immune cellsengrafts in the recipient subject. In one embodiment, the transplantedengineered immune cells reconstitute the immune system in the recipientsubject. The transfected cells can also be cryopreserved aftertransfected and stored, or cryopreserved after cell expansion andstored.

The engineered cells having multilineage hematopoietic progenitor cellsdescribed herein may be administered as part of a bone marrow or cordblood transplant in an individual that has or has not undergone bonemarrow ablative therapy. In one embodiment, genetically modified cellscontemplated herein are administered in a bone marrow transplant to anindividual that has undergone chemoablative or radioablative bone marrowtherapy.

In one embodiment, a dose of cells is delivered to a subjectintravenously. In one embodiment, the cells are intravenouslyadministered to a subject.

In particular embodiments, patients receive a dose of the cells, e.g.,engineered cells of this disclosure, of about 1×10⁵ cells/kg, about5×10⁵ cells/kg, about 1×10⁶ cells/kg, about 2×10⁶ cells/kg, about 3×10⁶cells/kg, about 4×10⁶ cells/kg, about 5×10⁶ cells/kg, about 6×10⁶cells/kg, about 7×10⁶ cells/kg, about 8×10⁶ cells/kg, about 9×10⁶cells/kg, about 1×10⁷ cells/kg, about 5×10⁷ cells/kg, about 1×10⁸cells/kg, or more in one single intravenous dose.

In certain embodiments, patients receive a dose of the cells, e.g.,engineered cells, of at least 1×10⁵ cells/kg, at least 5×10⁵ cells/kg,at least 1×10⁶ cells/kg, at least 2×10⁶ cells/kg, at least 3×10⁶cells/kg, at least 4×10⁶ cells/kg, at least 5×10⁶ cells/kg, at least6×10⁶ cells/kg, at least 7×10⁶ cells/kg, at least 8×10⁶ cells/kg, atleast 9×10⁶ cells/kg, at least 1×10⁷ cells/kg, at least 5×10⁷ cells/kg,at least 1×10⁸ cells/kg, or more in one single intravenous dose.

In an additional embodiment, patients receive a dose of the cells, e.g.,engineered cells of this disclosure, of about 1×10⁵ cells/kg to about1×10⁸ cells/kg, about 1×10⁶ cells/kg to about 1×10⁸ cells/kg, about1×10⁶ cells/kg to about 9×10⁶ cells/kg, about 2×10⁶ cells/kg to about8×10⁶ cells/kg, about 2×10⁶ cells/kg to about 8×10⁶ cells/kg, about2×10⁶ cells/kg to about 5×10⁶ cells/kg, about 3×10⁶ cells/kg to about5×10⁶ cells/kg, about 3×10⁶ cells/kg to about 4×10⁸ cells/kg, or anyintervening dose of cells/kg.

In general, the engineered cells described herein are administered as asuspension with a pharmaceutically acceptable carrier. For example, astherapeutic compositions. Therapeutic compositions contain aphysiologically tolerable carrier together with the cell composition andoptionally at least one additional bioactive agent as described herein,dissolved or dispersed therein as an active ingredient. In a preferredembodiment, the therapeutic composition is not substantially immunogenicwhen administered to a mammal or human patient for therapeutic purposes,unless so desired. One of skill in the art will recognize that apharmaceutically acceptable carrier to be used in a cell compositionwill not include buffers, compounds, cryopreservation agents,preservatives, or other agents in amounts that substantially interferewith the viability of the cells to be delivered to the subject. Aformulation comprising cells can include e.g., osmotic buffers thatpermit cell membrane integrity to be maintained, and optionally,nutrients to maintain cell viability or enhance engraftment uponadministration. Such formulations and suspensions are known to those ofskill in the art and/or can be adapted for use with the cells asdescribed herein using routine experimentation.

In some embodiments, the compositions of engineered cells describedfurther comprise a pharmaceutically acceptable carrier. In oneembodiment, the pharmaceutically acceptable carrier does not includetissue or cell culture media.

In various embodiments, a second or subsequent dose of cells isadministered to the recipient subject. For example, second andsubsequent administrations can be given between about one day to about30 weeks after the previous administration. Two, three, four or moretotal administrations can be delivered to the individual, as needed.

A cell composition can be administered by any appropriate route whichresults in effective cellular replacement treatment in the subject, i.e.administration results in delivery to a desired location in the subjectwhere at least a portion of the composition delivered, i.e. at least1×10⁴ cells are delivered to the desired site for a period of time.Modes of administration include injection, infusion, or instillation,“Injection” includes, without limitation, intravenous, intra-arterial,intraventricular, intracardiac injection and infusion. For the deliveryof cells, administration by injection or infusion is generallypreferred.

Efficacy testing can be performed during the course of treatment usingthe methods described herein. Measurements of the degree of severity ofa number of symptoms associated with a particular ailment are notedprior to the start of a treatment and then at later specific time periodafter the start of the treatment.

The embodiments of the present disclosure can be defined in any of thefollowing numbered paragraphs:

-   -   1. A method for making hematopoietic stem cells (HSCs) and        hematopoietic stem and progenitor cells (HSPCs) comprising in        vitro transfecting hemogenic endothelia cells (HE) with an        exogenous gene coding copy of at least one of the following        transcription factors ERG, HOXA9, HOXA5, LCOR and RUNX1        comprised in a non-integrative vector, wherein the transcription        factors are expressed in the transfected cells to produce a        population of multilineage HSCs and HSPCs that engrafts in        recipient host after implantation.    -   2. A method of making hematopoietic stem cells (HSCs) and        hematopoietic stem and progenitor cells (HSPCs) comprising:        -   a. generating embryonic bodies (EB) from pluripotent stem            cells;        -   b. isolating hemogenic endothelia cells (HE) from the            resultant population of EB;        -   c. inducing endothelial-to-hematopoietic transition (EHT) in            culture in the isolated HE to obtain hematopoietic stem            cells, and        -   d. in vitro transfecting the induced HE with an exogenous            gene coding copy of at least one of the following            transcription factors ERG, HOXA9, HOXA5, LCOR and RUNX1            comprised in a non-integrative vector.    -   3. The method of paragraphs 1 or 2, wherein the method is an in        vitro method.    -   4. The method of any one of paragraphs 1-3, wherein the EB are        generated or induced from pluripotent stem cells (PSC) by        culturing or exposing the PSC to mophogens for about 8 days.    -   5. The method of paragraph 4, wherein the mophogens selected        from the group consisting of Holo-Transferrin, mono-thioglycerol        (MTG), ascorbic acid, bone morphogenetic protein (BMP)-4, basic        fibroblast growth factor (bFGF), SB431542, CHIR99021, vascular        endothelial growth factor (VEGF), interleukin (IL)-6,        insulin-like growth factor (IGF)-1, interleukin (IL)-11, stem        cell factor (SCF), erythropoietin (EPO), thrombopoietin (TPO),        interleukin (IL)-3, and Fms related tyrosine kinease 3 ligand        (Flt-3L).    -   6. The method of any one of paragraphs 1-5, wherein the EBs are        less than 800 microns in size and are selected.    -   7. The method of any one of paragraphs 1-6, wherein the EB cells        within the EBs are compactly adhered to each other and requires        trypsin digestion in order to dissociate the cells to individual        cells.    -   8. The method of paragraph 6 or 7, wherein the EB cells of the        selected EBs are dissociated prior to the isolation of HE.    -   9. The method of any one of paragraphs 1-8, wherein the        population of PSC is induced pluripotent stem cells (iPS cells)        or embryonic stem cells (ESC).    -   10. The method of paragraph 9, wherein the induced pluripotent        stem cells are produced by introducing only reprogramming        factors OCT4, SOX2, KLF4 and optionally c-MYC or nanog and LIN28        into mature cells.    -   11. The method of paragraph 10, wherein the mature cells are        selected from the group consisting of B lymphocytes (B-cells), T        lymphocytes, (T-cells), fibroblasts, and keratinocytes.    -   12. The method of paragraph 8, 9 or 10, wherein the induced        pluripotent stem cells are produced by introducing the        reprogramming factors two or more times into the mature cells.    -   13. The method of any one of paragraphs 1-12, wherein the HE are        definitive HE.    -   14. The method of any one of paragraphs 1-13, wherein the HE are        isolated immediately from selected and dissociated EB.    -   15. The method of any one of paragraphs 1-14, wherein the HE are        FLK1+, CD34+, CD43−, and CD235A−. (these biomarkers are those on        HE before the endothelial-to-hematopoietic transition?)    -   16. The method of any one of paragraphs 1-15, wherein the        hematopoietic cells are CD34+ and CD45+.    -   17. The method of any one of paragraphs 1-16, wherein the        endothelial-to-hematopoietic transition occurs by culturing the        isolated HE in thrombopoietin (TPO), interleukin (IL)-3, stem        cell factor (SCF), IL-6, IL-11, insulin-like growth factor        (IGF)-1, erythropoietin (EPO), vascular endothelial growth        factor (VEGF), basic fibroblast growth factor (bFGF), bone        morphogenetic protein (BMP)4, Fms related tyrosine kinase 3        ligand (Flt-3L), sonic hedgehog (SHH), angiotensin II, chemical        AGTR1 (angiotensin II receptor type I) blocker losartan        potassium.    -   18. The method of any one of paragraphs 1-17, wherein the        multilineage HSCs are CD34+CD38−CD45+.    -   19. The method of any one of paragraphs 1-18, wherein the        multilineage HSPCs are CD34+CD45+.    -   20. The method of paragraph 1 or 2, wherein the non-integrative        vector is an episomal vector.    -   21. The method of paragraph 1 or 2, wherein at least 2, at least        3, at least 4, or at least 5 transcription factors are        transfected.    -   22. An engineered cell derived from a population of HE and        produced by a method of any one of paragraphs 1-21.    -   23. The engineered cell of paragraph 22, wherein the engineered        cell comprises an exogenous copy of each of the following        transcription factors ERG, HOXA9, HOXA5, LCOR and RUNX1.    -   24. The engineered cell of paragraph 22 or 23, wherein the        engineered cell further comprises an exogenous copy of each of        the following reprogramming factors OCT4, SOX2, KLF4 and        optionally c-MYC.    -   25. A composition comprising a population of engineered cells of        any one of paragraphs 22-24.    -   26. The composition of paragraph 25, further comprising a        pharmaceutically acceptable carrier.    -   27. A pharmaceutical composition comprising a population of        engineered cells of any one of paragraphs 22-24 and a        pharmaceutically acceptable carrier.    -   28. A pharmaceutical composition of paragraph 27 for use in        cellular replacement therapy in a subject.    -   29. A method of cellular replacement therapy in a subject in        need thereof, the method comprising administering a population        of engineered cells of paragraphs 22-24, or a composition of        paragraph 25-26, or a pharmaceutical composition of paragraph 27        to a recipient subject.    -   30. The method of cellular replacement therapy of paragraph 29,        wherein the subject is a patient who has undergone chemotherapy        or irradiation or both, and manifest deficiencies in immune        function or lymphocyte reconstitution or both deficiencies in        immune function and lymphocyte reconstitution.    -   31. The method of cellular replacement therapy of paragraph 29        or 30, wherein the subject prior to implantation, the immune        cells are treated ex vivo with prostaglandin E2 and/or        antioxidant N-acetyl-L-cysteine (NAC) to promote subsequent        engraftment in a recipient subject.    -   32. The method of cellular replacement therapy of paragraph 29        or 30 or 31, wherein the immune cells are autologous to the        recipient subject or at least HLA type matched with the        recipient subject.

The embodiments of the present disclosure are further illustrated by thefollowing example which should not be construed as limiting. Thecontents of all references cited throughout this application, as well asthe figures and table are incorporated herein by reference.

Those skilled in the art will recognize, or be able to ascertain usingnot more than routine experimentation, many equivalents to the specificembodiments of the present disclosure described herein. Such equivalentsare intended to be encompassed by the following claims.

EXAMPLE Materials and Methods

hPSC Culture

Example 1

Material and Methods

hPSC Culture.

All experiments were performed with H9 hESC (NIHhESC-10-0062), PB34iPS⁵⁹, MSC-IPS1⁶⁰, PB34 iPS⁶⁵, MSC-iPS1⁶⁶, 1045-iPSC, and 1157-iPSCestablished by hES (human embryonic stem cell) core facility at BostonChildren's Hospital. See the Children's Hospital webpage at stemcellperiod childrenshospital period org. Human embryonic stem cells (ESCs)and induced pluripotent stem cells (iPSCs) were maintained on mouseembryonic fibroblasts (GLOBALSTEM) feeders in DMEM/F12+20% KNOCKOUTSERUMReplacement (INVITROGEN), 1 mM L-glutamine, 1 mM NEAA, 0.1 mMbeta-mercaptoethanol, and 10 ng/ml bFGF on 10 cm gelatinized culturedish. Media was changed daily, and cells were passaged 1:4 onto freshfeeders every 7 days using standard clump passaging with collagenase IV.Morphology of pluripotent stem cells (PSCs) was checked under microscopydaily. As a quality control, only dishes with more than 70% of typicalPSC colonies were processed for embryoid bodies (EBs) formation. EBs arethree-dimensional aggregates of pluripotent stem cells. Cell lines weretested for mycoplasma routinely.

EB Differentiation.

EB differentiation was performed as previously described¹⁹. Briefly,human pluripotent stem cells (hPSCs) colonies were dissociated with0.05% Trypsin for 5 min in at 37° C., pipetted thoroughly with p1000until single cells or small aggregates, washed twice with PBS with 2%fetal bovine serum (FBS), and re-suspended in StemPro-34-based mediaprior to seeding. StemPro-34 (INVITROGEN, 10639-011) is supplementedwith L-glutamine (2 mM), penicillin/streptomycin (10 ng/mL), ascorbicacid (1 mM), human holo-Transferrin (150 μg/mL, SIGMA T0665), andmonothioglycerol (MTG, 0.4 mM) (referred to as “SupplementedStemPro-34), BMP-4 (10 ng/mL) and Y-27632 (10 uM). The suspended cellswere seeded into non-adherent spheroid formation 10 cm plates(EZSPHERE™, ASAHI GLASS CO; Well Size (μm) Diameter: 400-500, Depth:100-200; No. of well 14,000/dish) at a cell density of 2-5 millioncells/dish. About 24 h after seeding, bFGF (5 ng/mL) and BMP4 (10 ng/mL)were added to the media. At day 2 in culture, the developing EBs werecollected and re-suspended in supplemented StemPro-34 with SB431542 (6μM), CHIR99021 (3 μM), bFGF (5 ng/mL) and BMP4 (10 ng/mL). The formationof EBs were checked under microscopy at day 4 and the decision was madeto continue EB formation based on the size and morphology ofaggregations (quality control; >100 uM, compaction-like tight contact ofcells). At day 4, the media is replaced by supplemented StemPro-34 withVEGF (15 ng/mL) and bFGF (10 ng/mL). At day 6, the media is replaced bysupplemented StemPro-34 with bFGF (10 ng/mL), VEGF (15 ng/mL), IL-6 (10ng/mL), IGF-1 (25 ng/mL), IL-11 (5 ng/mL) and SCF (50 ng/mL). Cultureswere maintained in a 5% CO₂/5% O₂/90% N₂ environment. After 8 days inculture, the EBs are harvested and sorted for definitive HE via surfacemarkers.

HE Sorting.

To avoid potential damage with hydrodynamic pressure and contaminationthrough FACS (Elliot et al., 2009), for functional assay, the isolationof HE was carried out by MACS. Freshly dissociated EB cells (at day 8time point) were filtered through a 70 um filter and stained with CD34microbeads (MILTENYI) for 30 min at 4° C. CD34+ cells were isolated withLS columns (MILTENYI). Around 0.1-0.5×10⁶ cells were obtained per 10 cmdish of EB formation. A sample from each batch was analyzed by FACS tovalidate its purity of HE. The following antibodies or dyes are used inthe FACS: CD34 PE-Cy7 (8G12; BD), FLK1 AF647 (89106; BD),CD235a/Glycophorin (GLY)-A FITC (11E4B-7-6; Coulter), CD43 PE (1G10; BD)and DAPI. For accurate isolation of HE for expression profile bymicroarray and qRT-PCR, isolation of HE was carried out by FACS.Dissociated EBs (at day 8 time point) were re-suspended at 1-3×10⁶ per100 ml staining buffer (PBS+2% FBS). Cells were stained with a 1:50dilution of CD34 PE-Cy7 (8G12; BD), FLK1 AF647 (89106; BD),CD235a/Glycophorin (GLY)-A FITC (11E4B-7-6; Coulter), CD43 PE (1G10; BD)and DAPI for 30 min at 4° C. dark. All FACS sorting was performed on aBD FACS Aria II cell sorter using a 100 micrometer nozzle to avoidpotential damage to HE.

Microarray and CellNet Analysis of HE.

All the samples used for microarray analysis were FACS sorted. Thefollowing antibodies are used for identifying HE: CD34 PE-Cy7 (8G12;BD), FLK1 AF647 (89106; BD), CD235a/Glycophorin (GLY)-A FITC (11E4B-7-6;Coulter), CD43 PE (1G10; BD). Fetal liver hematopoietic stem cells(HSCs) were purchased from STEMCELL Technologies and stained with thefollowing antibodies for identifying HSC: CD38 PE-Cy5 (LS198-4-3;Clontech), CD34 PE-Cy7, and CD45 PE (HI30; BD). Between 10,000-50,000cells were sorted for each cell type for each replicate (n=2 or 3). TheRNAeasy Microkit (QIAGEN) was used to collect and prepare total RNA formicroarray analysis. The Ovation Picokit (NUGEN) was used forpre-amplification, where required. Gene expression profiling wasperformed on AFFYMETRIX 430 2.0 gene chips per standard protocol.Microarray data were analyzed per standard protocol usingR/Bioconductor. The classification and GRN of HE were analyzed byCellNet (Cahan et al., 2014). Briefly, it was reasoned that the extentto which a GRN is established in a sample is reflected in the expressionof the genes in the GRN such that the GRN genes should fall within arange of expression observed in the corresponding C/T in the trainingdata. This notion was formalized as a GRN status metric, defined incomparison to the complete training data set. The status of C/T GRN in aquery sample is defined as the weighted mean of the Z scores of thegenes in the GRN, where the Z score is defined in reference to theexpression distribution of each gene in a C/T. The GRN status score canbe weighted by the absolute expression level of each gene in a C/T sothat genes more highly expressed have more influence on the GRN status(default) and/or by the importance to the Random Forest classifier.

Endothelial-to-Hematopoietic Transition (EHT) Culture.

EBs were dissociated on day 8 by digestion with 0.05% Trypsin for 5 minat 37° C., pipetted thoroughly with p1000 pipette to generate a singlecell suspension and washed with PBS+2% FBS. Dissociated EBs wereimmediately processed for the isolation of HE. Unlike HSPCs in previousreport that used frozen batches¹⁷, HE delayed (approximately 2-3 days)recovery of morphology and growth once frozen, thus all the experimentswere done with freshly isolated HE. Thus, cells were re-suspended in 1mL of PBS+2% FBS and incubated with human CD34 MicroBead kit for 1 h(MILTENYL BIOTEC, 130-046-702). After incubation, cells were washed withPBS+2% FBS and isolated by magnetic cell isolation (MACS) using LScolumns (MILTENYL BIOTEC, 130-042-401). Sorted CD34+ cells werere-suspended in supplemented StemPro-34 media, containing Y-27632 (10uM), TPO (30 ng/mL), IL-3 (10 ng/mL), SCF (50 ng/mL), IL-6 (10 ng/mL),IL-11 (5 ng/mL), IGF-1 (25 ng/mL), VEGF (5 ng/mL), bFGF (5 ng/mL), BMP4(10 ng/mL), and Flt-3L (10 ng/mL) as reported²⁰ and seeded at a densityof 25-50×10⁴ cells per well onto thin-layer MATRIGEL-coated 24-wellplates. All recombinant factors are human and most were purchased fromPeprotech.

Sorted HE cells were seeded onto thin-layer MATRIGEL-coated 96-wellplates (flat-bottom) at a density of 5×10⁴/well in supplementedStemPro-34 media, containing TPO (30 ng/ml), IL-3 (30 ng/ml), SCF (100ng/ml), IL-6 (10 ng/ml), IL-11 (5 ng/ml), IGF-1 (25 ng/ml), EPO (2U/ml), VEGF (5 ng/ml), bFGF (5 ng/ml), BMP4 (10 ng/ml), Flt-3L (10ng/ml), SHH (20 ng/ml), angiotensin II (10 μg/l) and the chemical AGTR1(angiotensin II receptor type I) blocker losartan potassium (100 μM,Tocris) as reported²⁰. All recombinant factors are human and most werepurchased from Peprotech. The exception is angiotensin II, which waspurchased from Sigma.

Lentivirus Production.

Plasmids for the TF library were obtained as Gateway plasmids (HarvardPlasmid Serive; GeneCopoeia). Open reading frames were cloned intolentiviral vectors using LR Clonase (INVITROGEN). Two vectors were used,pSMAL-GFP (constitutive) and pINDUCER-21⁶¹. Lentiviral particles wereproduced by transfecting 293T-17 cells (ATCC) with the second-generationpackaging plasmids (pMD2.G and psPAX2 from Addgene). Virus was harvested36 and 60 hr after transfection and concentrated by ultracentrifugationat 23,000 rpm for 2 hr 15 min at 4° C. Virus was reconstituted with 50uL of EHT culture media. Constructs were titered by serial dilution on293T cells using GFP as an indicator. Polycistronic vectors were made asfollows. LCOR-P2A-HOXA9-T2A-HOXA5 and RUNX1-P2A-ERG DNA fragments weresynthesized and cloned into TOPO-D cloning vector respectively byGENSCRIPT, then Gateway-recombined with pINDUCER-21.

Lentiviral Gene Transfer.

At day 3 of EHT culture, HE cells were beginning to produce potentiallyhematopoietic ‘round’ cells, the occurrence of this phenomenon used asquality control of HE induction and transition to hematopoietic cellsfor each batch of experiment. The infection media was EHT culture mediasupplemented with Polybrene (8 ug/mL, SIGMA). Lentiviral infections werecarried out in a total volume of 150 ul or 250 ul (for a 24-well plate).The multiplicity of infection for the factors was as follows: Library3.0 for each, ERG 5.0, HOXA5 5.0, HOXA9 5.0, HOXA10 5.0, LCOR 5.0, RUNX15.0, and SPI1 5.0. HE was vulnerable to damage during spinoculation,thus infections were carried out static for 12 hours, then 50 uL or 250uL of fresh EHT media was supplemented to dilute Polybrene. Parallelwells were kept cultured for additional 3 days to measure infectionefficiency by percentage of GFP+ DAPI-cells by FACS, 30-70% infectionefficiency.

In Vitro Screening Via CFU.

Followed by lentiviral gene transfer, cells were maintained for 5 daysin EHT culture media supplemented with doxycycline (2 ug/mL, SIGMA) toinduce transgene expression in vitro. 5×10⁴ cells were plated into 3 mlof complete methylcellulose (H4434; STEMCELL Technologies). Additionalcytokines added were: 10 ng/ml FLT3, 10 ng/ml IL6, and 50 ng/ml TPO (R&DSystems). The mixture was distributed into two 60 mm dishes andmaintained in a humidified chamber at 37° C. for 14 days. Colonies werescored manually or using the BD Pathway 855 fluorescent imager. At 14days, granurocyte/erythrocyte macrophage/mega-karyocyte (GEMM) colonieswere picked up by P20 pipette. Between 10-20 GEMM colonies were pickedfor each replicates (n=3). The QIAamp DNA Micro kit (QIAGEN) was used tocollect and prepare total genomic DNA for PCR detection of transgenes.Nested PCR reactions were: 1st round with LNCX Fwd primer (5′-AGC TCGTTT AGT GAA CCG TCA GAT C-3′) and EGFP N Rev primer (5′-CGT CGC CGT CCAGCT CGA CCA G-3′). This PCR program consists of 1 cycle of 95° C. for 5min, 36 cycles of 95° C. for 30 sec followed by 60° C. for 30 sec thenfollowed by 72° C. for 5 min, 1 cycle of 72° C. for 10 min, and aterminating cycle of 4° C. for indefinite period; 2nd round with forwardprimer for each gene (listed in Table) and HA Rev primer (5′-TCT GGG ACGTCG TAT GGG TA-3′). This PCR program consists of 1 cycle of 95° C. for 5min, 36 cycles of 95° C. for 30 sec followed by 60° C. for 30 sec thenfollowed by 72° C. for 30 sec, 1 cycle of 72° C. for 10 min, and aterminating cycle of 4° C. for indefinite period.

In Vivo Screening Via Transplantation.

12 hours after lentiviral gene transfer, cells were recovered by dispasefor 5 min at 37° C., and washed by PBS three times to ensure nocarry-over of virus. Cells were re-suspended at 0.5-1.0×10⁶ cells per 25uL buffer (PBS+2% FBS from STEMCELL Technologies) and kept on ice untilinjection. 0.5-1.0×10⁶ cells were intrafemorally injected toNOD/LtSz-scidlL2Rgnull (NSG) mice and treated with doxycycline asdescribed in the section on Mouse transplantation. Up to 100 uLperipheral blood (PB) were collected every 2-4 weeks, through 14 weeks.Mice were sacrificed and harvested for bone marrow (BM) and thymus at8-14 weeks. For transgene detection in engrafted cells, each lineagecells were FACS sorted from BM. The following antibodies are used foridentifying cell types, myeloid cells: CD33 APC (P67.6; BD), CD45 PE-Cy5(J33; Coulter). B-cells: CD19 PE (HIB19; BD), CD45 PE-Cy5 (J33;Coulter); T-cells: CD3 PE-Cy7 (SK7; BD), CD45 PE-Cy5 (J33; Coulter).Between 10,000-50,000 cells were isolated with 2 or 3 biologicalreplicates for multiple cell lines (iPSCs and ESCs). The QIAamp DNAMicro kit (QIAGEN) was used to collect and prepare total genomic DNA forPCR detection of transgenes. Nested PCR reaction was done as similar toin vitro screening of above section.

Mouse Transplantation.

NOD/LtSz-scidlL2Rgnull (NSG) (Jackson Labs) mice were bred and housed atthe Boston Children's Hospital (BCH) animal care facility. Animalexperiments were performed in accordance to institutional guidelinesapproved by BCH animal care committee. Intra-femoral transplants havebeen previously described. Briefly, 6-10 week old mice were irradiated(250 rads) 12 hrs before transplant. Prior to transplantation, mice weretemporarily sedated with isoflurane. A 27 g needle was used to drill theleft femur (injected femur), and 0.5-1.0×10⁶ cells were transplanted ina 25 μL volume using a 28.5 g insulin needle. Sulfatrim was administeredin drinking water to prevent infections after irradiation. 625 ppmDoxycycline Rodent Diet (Envigo-Teklad Diets) and Doxycycline (1.0mg/ml) was added to the drinking water to maintain transgene expressionin vivo for 2 weeks¹². Secondary transplantation was carried with1,000-2,000 human CD34+ cells (isolated from BM by MACS with CD34microbeads) at 8 weeks. Isolated cells were resuspended at 1,000-2,000cells per 25 uL buffer (PBS+2% FBS from STEMCELL Technologies) and kepton ice until injection. Cells were intrafemorally injected toNOD/LtSz-scidlL2Rgnull (NSG) and treated with Doxycycline via food anddrinking water for 2 weeks.

Flow Cytometry.

Cells grown in EHT culture or harvested animal tissues were stained withthe following antibody panels. HE panel: CD34 PE-Cy7 (8G12; BD), FLK1AF647 (89106; BD), CD235a/Glycophorin (GLY)-A FITC (11E4B-7-6; Coulter),CD43 PE (1G10; BD). HSPC panel: CD38 PE-Cy5 (LS198-4-3; Clontech), CD34PE-Cy7, and CD45 PE (HI30; BD). Lineage panel: CD235a/Glycophorin(GLY)-A PE-Cy7 or FITC (11E4B-7-6; Coulter), CD33 APC (P67.6; BD), CD19PE (HIB19; BD), IgM BV510 (G20-127; BD), CD4 PE-Cy5 (13B8.2; Coulter),CD3 PE-Cy7 (SK7; BD), CD8 V450 (RPA-T8; BD), TCRαβ BV510 (T10B9; BD),TCRγδ APC (B1; BD), CD45 PE-Cy5 (J33; Coulter), CD15 APC (HI98; BD) andCD31/PECAM PE (WM59; BD). All stains were performed with <1×106 cellsper 100 μl staining buffer (PBS+2% FBS) with 1:100 dilution of eachantibody, 30 min at 4C in dark. Compensation was performed by automatedcompensation with anti-mouse Igk negative beads (BD) and CB MNC stainedwith individual ab. All acquisition was performed on BD Fortessacytometer. For detection of engraftment, human cord blood-engraftedmouse marrow was used as a control to set gating; Sorting was performedon a BD FACS Aria II cell sorter.

Cytospin of Erythroid and Neutrophils.

5,000-10,000 of FACS sorted erythroid (CD235a/Glycophorin (GLY)-A PE-Cy7or FITC (11E4B-7-6; Coulter)), plasmacytoid lymphocytes (CD19 PE (HIB19;BD), IgM BV510 (G20-127; BD), CD38 PE-Cy5 (LS198-4-3; Clontech)) andneutrophils (CD15 APC (HI98; BD), CD31/PECAM PE (WM59; BD) and CD45PE-Cy5 (J33; Coulter)) were cytospun onto slides (500 rpm for 10minutes), air dried, and stained with May-Grunwald and Giemsa stains(both from Sigma), washed with water, air dried, and mounted, followedby examination by light microscopy.

Quantitative PCR.

RNA extraction was performed using the RNAeasy Microkit (QIAGEN).Reverse transcription was performed using Superscript III (>5,000 cells)or VILO reagent (<5,000 cells) (Invitrogen). Quantitative PCR wascarried out in triplicate with SYBR Green (Applied Biosystems).Transcript abundance was calculated using the standard curve method.Primers used for globin genes62: huHbB F (5′-CTG AGG AGA AGT CTG CCGTTA-3′) (SEQ ID NO: **), huHbB R (5′-AGC ATC AGG AGT GGA CAG AT-3′) (SEQID NO: **), huHbG F (5′-TGG ATG ATC TCA AGG GCA C-3′) (SEQ ID NO: **),huHbG R (5′-TCA GTG GTA TCT GGA GGA CA-3′) (SEQ ID NO: **), huHbE F(5′-GCA AGA AGG TGC TGA CTT CC-3′) (SEQ ID NO: **), huHbE R (5′-ACC ATCACG TTA CCC AGG AG-3′) (SEQ ID NO: **).

ELISA of Terminally Differentiated Cells.

FACS isolated neutrophils (CD15+PECAM+CD45+), T-cells (CD3+CD45+) werecultured in IMDM+10% FBS overnight in 96-well pate (flat-bottom). Thensupernatant was taken and analyzed by MPO- or IFNγ-ELISA-Ready-SET-Go!Kit (eBioscience) according to the manufacturer's protocol. The amountof IFNγ was normalized per 1,000 cells. Human Ig production was measuredfrom 50 uL of serum from NSG mice at 8 weeks (IgM) and 14 weeks postengraftment (IgG). Immunization of mice was done with OVA (F5503, Sigma)with Freund's complete adjuvant (F5881, Sigma), followed by boosterdoses of Freund's incomplete adjuvant (F5506, Sigma) according tomanufacture's instruction.

T-Cell Receptor CDR3 Sequencing.

Human CD3+ T cells were FACS-isolated from thymus of engrafted NSG mice.Purified DNA was subjected to next generation sequencing of thecomplementarity determining region 3 (CDR3) using immunoSEQ (AdaptiveBiotechnology, Seattle, Wash.) and analyzed with the immunoSEQ Analyzersoftware (Adaptive Biotechnology).

Affymetrix SNP 6.0 Genotyping of Engrafted Cells.

250 ng aliquots of genomic DNA from human CD45+ BM cells (CD45 PE-Cy5(J33; Coulter)) from engrafted NSG mice and original PSCs (2 biologicalreplicates) were digested with either Nsp1 or Sty 1. A universal adaptoroligonucleotide was then ligated to the digested DNAs. The ligated DNAswere diluted with water and three 10 uL aliquots from each well of theSty 1 plate and four 10 uL aliquots from each well of the Nsp1 platewere transferred to fresh 96-well plates. PCR master mix was added toeach well and the reactions cycled as follows: 94° C. for 3 min; 30cycles of 94° C. for 30 s, 60° C. for 45 s, 68° C. for 15 s; 68° C. for7 min; 4° C. hold. Following PCR, the 7 reactions for each sample werecombined and purified by precipitation from 2-propanol/7.5M ammoniumacetate. The UV absorbance of the purified PCR products was measured toinsure a yield ≥4 ug/uL. 45 uL (≥180 ug) of each PCR product wasfragmented with DNAse 1 so the largest fragments were <185 bp. Thefragmented PCR products were then end-labeled with a biotinylatednucleotide using terminal deoxynucleotidyl transferase. Forhybridization, the end-labeled PCR products were combined withhybridization cocktail, denatured at 95° C. for 10 min and incubated at49° C. 200 mL of each mixture was loaded on a GeneChip and hybridizedovernight at 50° C. and 60 rpm. Following 16-18 hrs of hybridization,the chips were washed and stained using the GenomeWideSNP6_450 fluidicsprotocol with the appropriate buffers and stains. Following washing andstaining, the GeneChips were scanned on a GeneChip Scanner 3000 usingAGCC software. Genotype calls (chp files) were generated in AffymetrixGenotyping Console using the default parameters. The resulting chp fileswere analyzed for familial relationships using the identity by statealgorithm implemented in Partek Genomics Suite.

Pooled RNA-Sequencing.

Engrafted human CD34+CD38−CD45+HSCs were isolated from BM from eitheriPS-derived HE- or CB-injected NSG mice, then RNA was purified withRNeasy Micro kit (QIAGEN). QC of RNA was done by Bioanalyzer and Qubitanalysis. Passed samples were converted into library and sequenced byNextseq PE75 kit.

Single-Cell RNA-Sequencing with in-Droplet-Seq Technology.

Engrafted human CD34+CD38−CD45+HSCs were isolated from BM from eitheriPS-derived HE- or CB-injected NSG mice, then processed forin-droplet-barcoding according to a previous report⁶³. Library was QCedwith Bioanalyzer and sequenced by Nextseq PE 75 kit.

Lentiviral Integration Detection by Ligation-Mediated PCR andNext-Generation Sequencing.

Either CD33+myeloid, CD19+ B- and CD3+ T-cells were isolated from BMfrom HE-injected NSG mice. Genomic DNA was purified with QIAamp DNAMicro kit (QIAGEN). Ligation-mediated PCR-based detection of lentiviralintegration sites was done with Lenti-X Integration Site Analysis Kit(Clontech) according to manufacture's instruction. Sequencing-baseddetection (Integ-seq) was done following a previous report⁶⁴.

Single-Cell RNA-Sequencing with in-Droplet-Seq Technology.

Engrafted human CD34+CD38−CD45+HSCs were isolated from BM from eitheriPS-derived HE- or CB-injected NSG mice, then processed forin-droplet-barcoding according to a previous report 72. Library was QCedwith Bioanalyzer and sequenced by Nextseq PE 75 kit. The t-DistributedStochastic Neighbor Embedding (t-SNE) algorithm was used to visualizetranscriptome similarities and population heterogeneity of cord bloodHSCs and iPSC-derived HSCs. t-SNE performs a dimensionality reduction ofmultidimensional single-cell RNA-seq data into a low dimensional space,preserving pairwise distances between data-points as good as possible,allowing a global visualization of subpopulation structure and cell-cellsimilarities. The R package tsne was used in the analyses presentedherein. The t-SNE map was initialized with point-to-point distancescomputed by classical multidimensional scaling and the R plot functionwas used to visualize t-SNE maps annotated by cord blood or iPSC-derivedHSCs. Plots showing t-SNE maps colored by expression of selected geneswere created using the ggplot2 package. For subpopulationidentification, the top 500 genes with highest variance were used toelucidate global differences among single cells. To assess transcriptomesimilarities in terms of induction of hematopoietic genes iniPSC-derived HSCs, 62 hematopoietic genes were used for t-SNE analysis.

GSEA.

GSEA was performed with the desktop client version (javaGSEA, softwareis available at the Broad Institute website) with default parameters.RPKM values from the 7F-HSPC were obtained from the RNA seq (describedpreviously). These values were normalized to a terminally differentiatedcell (e.g., T-cells or B-cells) and the normalized values were used torank the most differentially expressed genes. These differentiallyexpressed genes were used to run GSEA with gene sets obtained frommSigDB (KEGG, Hallmark, immunological, transcription factors andchemical and genetic perturbations gene sets were used). In addition,gene sets specific to progenitors, cord blood or fetal-liver HSC wereobtained from previous reports⁷³ 2³. FDR <0.25 with a p-value of <0.05was considered significant.

Lentiviral Integration Detection by Ligation-Mediated PCR andNext-Generation Sequencing.

Either CD33+myeloid, CD19+ B- and CD3+ T-cells were isolated from BMfrom HE-injected NSG mice. Genomic DNA was purified with QIAamp DNAMicro kit (QIAGEN). Ligation-mediated PCR-based detection of lentiviralintegration sites was done with Lenti-X Integration Site Analysis Kit(Clontech) according to manufacture's instruction. Sequencing-baseddetection (Integ-seq) was done following a previous report⁷⁴.

Results

Cell identity is defined by gene regulatory networks that are governedby transcription factors (TFs)^(10,11). By supplying TFs that drivehematopoietic gene regulatory networks, several groups have generatedhematopoietic stem and progenitor cells from sources as diverse asfibroblasts, endothelial cells, and differentiated blood cells¹²⁻¹⁸. Ina prior study, a set of 9 HSC-specific TFs were screened for theirpotential to induce in vitro hematopoietic colony forming activity andin vivo engraftment from hPSC-derived myeloid cells, and isolated a setof five TFs (HoxA9, ERG, RORA, SOX4, and MYB) that promoted short-termengraftment of erythroid and myeloid cells, but did not achievelong-term multilineage hematopoiesis¹⁷. Recent approaches haverecapitulated HE differentiation from hPSCs to generate cells withmyeloid and T cell hematopoietic potential in vitro¹⁹⁻²¹; however, few,if any, cells capable of engrafting irradiated murine hosts weregenerated.

A protocol was adapted to derive HE from hPSCs and verified itshematopoietic potential²⁰. HE (characterized by these markers:FLK1+CD34+CD43−CD235A−) were isolated at day 8 of embryoid body (EB)formation (data not shown), and upon further culture supplemented withhematopoietic cytokines observed the endothelial to hematopoietictransition. Consistent with previous reports¹⁹²⁰ a decrease inexpression of endothelial genes (YAP, FOXC1, COUPTFII) was documented,an increase in levels of hematopoietic lineage genes (RUNX1, MYB, GATA2,SCL), and concomitant emergence of CD34+CD45+hematopoietic cells (datanot shown). However, multiple attempts to engraft irradiated immunedeficient recipients with these cultured cells failed.

Without wishing to be bound by a particular theory, it was hypothesizedthat introduction of HSC-specific TFs would endow hPSC-derived HE withthe potential to engraft multi-lineage hematopoiesis in vivo. The HE wasqueried by CellNet, a cell-type classification algorithm that comparesin vitro derived cells against a panel of comparator cell types²². HEwas classified as predominantly endothelium with partial identity tohematopoietic stem and progenitor cells (HSPCs; data not shown). Toidentify TFs likely to specify HSPC fate, it was reasoned thatfunctionally relevant TFs would be evolutionarily conserved. Thus, twoindependent mouse^(23,24) and two human^(25,26) datasets were used toselect 12 TFs enriched in fetal liver-HSCs (FL-HSCs) relative to HE(data not shown), and selected other candidates from prior reports thatused TFs to covert endothelial cells¹³, hPSC-derived myeloid cells¹⁷, orcommitted lymphoid cells¹² to hematopoietic progenitor cells. For thedata set, comparison of the expression profile of HSC-specific TFsbetween HE (CD34+FLK+CD43−CD235A−) vs FL-HSCs (CD34+CD38−CD90+CD45+)were made. 12 HSC-specific TFs were enriched in FL-HSCs anddownregulated in HE. Those TFs were cloned to Dox-inducible lentiviralvector. The expression level of SOX17, a marker of HE, was 2.4-foldhigher in HE (N=7) than FL-HSCs (N=10). * P<0.001. All together, alibrary of 26 TFs was assembled, which were cloned into adoxycycline-inducible lentiviral vector (FIG. 1A). The library wasinfected into HE at day 3 of endothelial-to-hematopoietic transition(EHT) culture (efficiency was >50%; FIGS. 1A and 4). The transducedcells were injected intrafemorally into sublethally irradiatedimmune-deficient NOD/SCID/Gamma (NSG) mice. Mice received doxycycline intheir drinking water and diet for two weeks to induce transgeneexpression¹², after which doxycycline was withdrawn. Human CD45+ cellswere observed in peripheral blood of injected mice up to 14 weeks,indicating long-term hematopoietic engraftment (FIG. 1B). Examination ofBM and thymus demonstrated the presence of human erythroid (GLY-A+),myeloid (CD33+), B (CD19+) and T-cells (CD3+) (FIG. 1C). When human cordblood (CB)-HSCs engraft in NSG mice, a predominance of B-cells over Tcells is typical²⁷. Analysis of BM from 3 of 6 recipients engrafted withhiPSC-derived HE and 2 of 5 recipients engrafted with hESC-derived HE at10-14 weeks showed notable reconstitution of T-cells and/or B-cells,comprising 46±20% and 37±9.7% proportion of human grafts, compared withCB-HSC derived grafts, in which B-cells comprised 86+11% of engraftedhuman cells (FIG. 1C). Intrafemoral injection of HSCs into one femurrepopulates the contralateral femur through expansion and homing ofHSCs²⁸. Notably, following unilateral intrafemoral injection oftransduced HE, engraftment in both femurs was observed. Engraftment ofhuman CD45+ cells in BM averaged 2.5±3.4% from transduced HE, comparedwith 46±18% from CB-HSCs. To confirm the hPSC origin of the engraftedcells, it was demonstrated by SNP array genotyping that human CD45+cells collected from peripheral blood were identical to the input hPSCs(data not shown). Together, these results demonstrate that infectionwith a 26-TF library enables multi-lineage hematopoietic engraftmentfrom hPSC-derived HE.

It was then determined which of the 26 TFs could be detected in theengrafted human cells by PCR amplification in sorted populations ofCD33+myeloid, CD19+ B cell, and CD3+ T cells. 7 TFs (ERG, HOXA5, HOXA9,HOXA10, LCOR, RUNX1, SPI1) were consistently detected in myeloid, B- andT-cells of five engrafted recipients, indicating that these factorsconferred multipotency. FIG. 5 shows the Venn diagram of expressionprofile of TFs during hematopoietic ontogeny. ZKSCAN1, SSBP2, MAFF,DACH1, and SOX4 were detected in certain infected cultures but notconsistently across all animals, perhaps reflecting their potential toenhance engraftment under some experimental conditions, or perhapsindicating that these genes are passengers.

It was next determined whether the 7 common TFs were sufficient tosupport multilineage engraftment of HE in vivo. HE with these 7 TFs weretransduced, injected cells intrafemorally into sublethally irradiatedNSG mice, and treated with doxycycline for 2 weeks. Chimerism of humanCD45+ in murine BM at 8 weeks was 1.9±1.8% for library transduced cells,12±5.1% for cells transduced with the defined 7 TFs, and 43±4.2% forCB-HSCs (FIGS. 1B and 1D), reflecting considerably enhanced engraftmentpotential for the 7TFs. It was sought to determine the minimalcombination of TFs required for multilineage engraftment by afactor-minus-one (FMO) approach. Exclusion of individual factors did notablate engraftment, though RUNX1, ERG, LCOR, HOXA5 or HOXA9 omissionreduced chimerism in BM most significantly at 8 weeks (FIG. 2B). Thesedata indicate that at a minimum, RUNX1, ERG, LCOR, HOXA5 and HOXA9facilitate optimal engraftment.

Mice engrafted with HE transduced with the defined 7 TFs were monitoreduntil 12 weeks. 2 out of 5 recipients had multi-lineage engraftment witherythroid (GLY-A+), myeloid (CD33+), B-cells (CD19+) and T-cells (CD3+)(FIGS. 1D and 1F). The 3 other recipients had both B-cells and T-cellsand either erythroid or myeloid cells (FIG. 1F). The self-renewalcapacity of HE-derived cells was next validated by secondarytransplantation. 2 out of 5 recipients engrafted with multilineageerythroid, neutrophils, B-cells and T-cells at 8 weeks (FIG. 2C). Thepercentage of phenotypic HSCs (CD34+CD38−) was lower in secondary thanprimary mice, indicating depletion of the HSC-like population over time,however, multilineage engrafts were still observed over 14 weeks (FIG.2D).

To determine which of 7 TFs were consistently isolated in multiplelineages of secondary engrafted mice, PCR amplification of myeloid, Band T cells was performed from 2 mice and detected LCOR, HOXA5, HOXA9and RUNX1 in every lineage, while ERG was noted in only myeloid andB-cells. SPI1 and HOXA10 appeared dispensable (FIG. 2A). IT was theninvestigated if ERG, LCOR, HOXA5, HOXA9 and RUNX1 are essential toconfer functional hematopoiesis on iPS-HE. 5 factors(LCOR-HOXA5-HOXA9/RUNX1-ERG) were induced by polycistronic lentiviralvectors and conferred multilineage erythroid, neutrophils, B-cells(including plasmacytoid lymphocytes) and T-cells at 12 weeks (FIGS. 1Eand 1F).

The RUNX1 TF is well known to facilitate EHT²⁹. It was determined ifdefined TFs enhance EHT using the RUNX1+24 enhancer-tdTomato reporterthat activates during hematopoietic cell emergence from HE³⁰. Uponexpression of 7 TFs in HE, the reporter was induced 2.4-fold compared tocontrol, correlating with an increase in hematopoietic genes (MYB,HDAC1, GATA2) (data not shown). These data indicated that the 7 TFsdrive hematopoiesis in part through facilitating Runx1-mediated EHT.

A previous report demonstrated the induction of engraftable progenitorcells from hESCs, however, it is uncertain if myeloid and lymphoidprogeny came from monoclonal origin that is feature of stem cells, ordistinctly committed progenitor cells¹⁸. It was then determined whetherthe myeloid and lymphoid progeny of the iPS-derived HE transduced withthe 7 TFs were of monoclonal origin by comparing lentiviral integrationpatterns. Genomic DNA was isolated from myeloid (CD33+), B-(CD19+) andT-cells (CD3+) from BM and thymus at 10 weeks post transplantation,followed by adaptor-ligation PCR to detect lentiviral integrationsequences. Common integration sequences were detected in myeloid,B-cells, and T-cells in each individual recipient (data not shown),consistent with a monoclonal origin of at least some of the cells.Remarkably, T-cells had several unique minor integrations indicating thepresence of a diverse human T-cell pool in engrafted NSG, as reportedpreviously³¹. These data demonstrate that in vivo screening identified acore set of between 5-7 TFs that induce HSC-like cells capable ofrepopulating irradiated primary and secondary mice with multi-lineagehematopoiesis.

Although some recipient mice manifest clonal multi-lineage hematopoiesisindicative of reconstitution from HSC-like cells, secondary mice showeda reduced frequency of CD34+38-phenotypic HSCs. The frequency ofphenotypic HSCs in BM was 2.0% (CB-HSC engrafted group) versus 0.47% (HEengrafted group) (FIG. 2F). The cycling state of 7 TF HSPCs (based onKi67 expression) was significantly higher than that of CB-HSCs insecondary recipients (FIG. 2G). Human CD45+ cells recovered fromengrafted BM revealed residual transgene expression despite cessation ofdoxyxcyline (data not shown). Interestingly, LCoR has been reported tobe a negative regulator of p21 expression³², and p21-deficient HSCs areknown to undergo exhaustion due to persistent cycling³³. Thus, tracelevels of residual transgene expression compromise the long term cyclingof the HSPC-like cells.

Erythroid, myeloid, and lymphoid cells recovered from engrafted micewere examined, and compared their functional properties toCB-HSC-derived cells. Definitive erythropoiesis is characterized byglobin switching and enucleation³⁴ ³⁵. Most erythroid cells generatedfrom hPSCs express embryonic and fetal globins and retain nuclei¹⁷. Thehuman erythroid cells recovered from engrafted mice lacked expression ofembryonic HBE, and expressed fetal HBG and adult HBB at levelscomparable to CFU-E from human CB (FIG. 3A). Remarkably, 1/4 of humanGLY-A+ cells derived from the 7TF HSPCs were enucleated (FIG. 3B). Humanmyeloid cells in NSG recipients respond to cytokine stimuli byactivation of myeloperoxidase (MPO)³⁶. CD45+CD15+PECAM+neutrophils wereisolated from engrafted BM at 8 weeks, and compared MPO production toengrafted CB-derived cells. PMA stimulation enhanced the production ofMPO 3.0-fold relative to unstimulated neutrophils (FIG. 3C). Bona fidehuman HSCs generate functional T- and B-cells in NSG mice²⁷. IgM and IgGantibody could be detected in the serum of NSG mice engrafted with 7 TFHSPCs, indicating the cooperative activity of T and B cells in mediatingimmunoglobulin class switching, secretion and boosted by Ovalbumin (OVA)(FIGS. 3D and 3E). mature CD3+ T-cells from BM were isolated andinterferon γ (IFNγ) production was measured. Notably, this CD3+population expressed CD4, and not CD8. PMA/Ionomycin (PMAI)-stimulationenhanced IFNγ production 3.0-fold in 7 TF CD3+ cells vs 4.4-fold inCB-derived CD3+(FIG. 3F). T-cells develop from CD4−CD8− double-negativecells followed by CD4+CD8+double-positive cells that express surfaceTCR/CD3 complex, which differentiate to either CD4 or CD8single-positive T cells in thymus, which migrate to blood and BM37. At 8weeks, 7 TF HSPC-derived thymocytes were predominantly CD4+CD8+(55±22%),with few CD4+CD8− (1.8±0.42%) and CD4-CD8+(0.80±0.36%). Human CD3+T-cells differentiated from HSCs in NSG possess either TCRαβ (>60%) orTCRγ6 (<30%)²⁷, consistent with the observation that 7 TF HSPC-derivedgrafts produced both TCRαβ (89±9.3%) and TCRγ6 (3.8±6.6%) cells (FIG.3G). Development of a diverse population of antigen-specific T cellsrequires rearrangement of germline-encoded TCR genes³⁸, largely mediatedby the complementarity determining region 3 (CDR3) within variable (V)gene segments of the TCRA and TCRB genes³⁹. To determine clonotypediversity, the CDR3 region of TCRB on CD3+ T-cells was profiled inreconstituted mice using next generation sequencing. A high degree ofcombinatorial diversity was observed in the V gene segment in CD3+T-cells isolated from either CB-engrafted NSG or 7 TF HSPC-engrafted NSGmice with the CDR3 length following a standard Gaussian distribution(FIG. 3H).

The generation of functional HSC-like cells from PSCs has been a longsought goal in hematology research. By directed differentiation of hPSCsto HE followed by in vivo screening of TFs for hematopoietic progenitorspecification, 7 TFs that together confer HSC-like engraftment,self-renewal and multilineage capacity were identified. Considerablework remains to establish engraftment with transgene-free cells, and toachieve stable, long-term multi-lineage hematopoietic chimerism inhumanized mice. Such a system will facilitate modeling a multitude ofgenetic blood disorders that are not faithfully recapitulated ingenetically engineered murine models, and for which adequate marrowsamples are not readily obtained, as for patients with bone marrowfailure syndromes.

Combinations of TFs have been introduced into differentiated bloodcells¹² ¹⁷ to endow HSC-like properties in a murine system¹², buttransplantable human HSCs with multilineage capacity have to date notbeen derived from hPSCs. Recent advances in the directed differentiationof PSCs to definitive HE have provided a supportive context forscreening of HSC-specifying TFs. Each of the identified TFs in the studypresented herein plays a role in HSC development, maintenance oflong-term HSCs, or lineage commitment. RUNX1 is essential forhematopoietic commitment of HE and can convert endothelial cells tohematopoietic progenitor cells¹³ ⁴⁰ ⁴¹. LCOR, a component of histonedeacetylation complex, is mutated in B-cell lymphoma, indicating a rolein B-lymphopoiesis⁴² ⁴³, but this factor has not previously beenimplicated in HSC functions and its role remains to be defined. SPI1(also known as PU. 1) is required for hematopoietic progenitor cellemergence and regulates myeloid specification⁴⁴ ⁴⁵ ⁴⁶. HOX familymembers have been reproducibly implicated in hematopoiesis acrossspecies^(17,47,48). HOXA9 is the key homeotic gene that defines HSCidentity^(49,50), interacting with ERG to support HSC renewal duringembryogenesis and stress hematopoiesis⁵¹⁻⁵³, indicating a basis for thefunctional cooperation of HOXA9 and ERG in the system presented herein.HOXA10 augments induction of erythroid cells from ESCs⁵⁴, but appears tobe at least partially dispensable in the system presented herein.Ectopic expression of HOXA5 induces commitment of HSCs to myeloidlineages⁵⁵. HOXA5 is a transcriptional target of Notch signaling inT-cell progenitors along with HOXA9 and HOXA10, consistent with a rolein T-lymphopoiesis⁵⁶. These factors share binding sites in the genomeand cooperate to recruit chromatin modulators (e.g. RUNX1 and HOXAfamilies)^(53,57) to induce and maintain HSPCs.

The FMO approach presented herein to the defined 7 TFs indicated thatthey are part of a common gene regulatory network with some redundancy,as exclusion of individual factors did not fully abrogate engraftment ofHE. The possibility remains that 7 TF HSPCs are predominantly fetal asshown in hESC-derived hematopoietic cells in a previous study⁵⁸,supported by their rapid cycling state, and predominance of CD4+CD8+ Tcells in the thymus.

The study presented herein indicates a potential of derivation ofHSC-like cells from renewable sources like pluripotent stem cells. Suchcells, when derived from patients with genetic blood disorders, offerconsiderable promise for modeling human blood disease, for humanizingmice for research applications, and for testing the capacity of genetherapy vectors or pharmacologic agents to restore hematopoieticfunction. The long term goal remains the derivation of bona fidetransgene-free HSCs for applications in research and therapy.

The references cited herein and throughout the specification areincorporated herein by reference.

REFERENCES

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Example 2

Hematopoietic stem and progenitor cells (HSPCs) generated frompluripotent stem cells (PSCs) constitute a valuable resource formodeling human blood diseases, drug screening or cell therapies.Derivation of human HSPCs from PSCs requires integrative viral vectorsthat may alter cell differentiation and restrict their clinical use.Shown herein is the generation of human HSPCs using non-integratingepisomal vectors to transiently express transcription factors thatconvert PSC-derived hemogenic endothelium into HSPCs. It is demonstratedherein that these HSPCs maintain long-term multi-lineage engraftmentafter the loss of episomal plasmids, indicating faithful reprogrammingin the absence of sustained expression of exogenous transgenes. Alimiting-dilution transplantation assay in secondary mice revealed asimilar frequency of HSPCs in the CD34+ cells engrafted in primary micefrom PSCs as for umbilical cord blood (UBC). Finally, single-cell mRNAsequencing showed that mature blood cells derived from UBC and episomalHSPCs were markedly similar, supporting integration-free HSPCs fortranslational approaches.

Introduction

Treatment of genetic blood diseases by transplantation of allogeneichematopoietic stem cells (HSCs) can be curative, but wider use islimited by the lack of optimal donors and complications associated withimmune mismatch (Morgan et al., 2017). The possibility of usingautologous HSCs derived from human pluripotent stem cells (hPSCs) orpatient-derived somatic cells would overcome these limitations. It isknown that cell fate conversion can be achieved by the ectopicexpression of transcription factors (TFs) (Cahan et al., 2014; Ivanovset al., 2017). In this regard, hematopoietic cells have been generatedby conversion of somatic cell types such as fibroblasts, endothelium, orrespecification along distinct lineages of differentiated blood cellsthrough the induction of specific combinations of TFs (Batta et al.,2014; Doulatov et al., 2013; Elcheva et al., 2014; Pereira et al., 2013;Riddell et al., 2014; Sandler et al., 2014; Szabo et al., 2010) ENREF38. Until recently, these strategies have not been successful for thegeneration of functional human HSCs, presumably due to a lack ofsufficient knowledge of the full regulatory complexities of humanhematopoiesis and a limited understanding of developmental trajectorieswithin the hematopoietic system (Guibentif and Gottgens, 2017; Lummertzda Rocha et al., 2018). ENREF 25

Several groups have now achieved the derivation in vitro ofhematopoietic stem and progenitor cells (HSPCs) with in vivo engraftmentpotential (Lis et al., 2017; Sugimura et al., 2017; Tan et al., 2018;Tsukada et al., 2017). It is now well-known that HSCs derive fromhemogenic endothelial (HE) cells (Bertrand et al., 2010; Boisset et al.,2010; Chen et al., 2011; de Bruijn et al., 2002; Dieterlen-Lievre, 1975;Dzierzak and Speck, 2008; Ivanovs et al., 2017; Ivanovs et al., 2011).Using lentiviral transduction, work described herein demonstrated thatfive transcription factors (5TFs) (LCOR, HOXA9, HOXA5, RUNX1 and ERG)are sufficient to drive conversion of HE cells into HSPCs withmulti-lineage and self-renewal engraftment capacities (Sugimura et al.,2017). This successful strategy combines morphogen-directeddifferentiation aimed at recapitulating human HSC specification in vitrowith conversion mediated by TF induction (Sugimura et al., 2017).However, the doxycycline (DOX)-inducible lentiviral vectors used foractivating the expression of the 5TFs integrate into the cell's genome,potentially leading to insertional mutations and limiting the capacityof these cells to undergo terminal maturation (Okita et al., 2007; Yu etal., 2007). To overcome these limitations, a strategy for transgeneexpression from non-integrating episomes was employed to drive HSPCproduction from human PSCs. Data presented herein demonstrate that fullyreprogrammed HSPCs can be achieved through transient expression of thepreviously established 5TF combination, thereby laying a foundation forapplications in research and therapy.

Results

Episomal-5TF-Derived HSPCs Show Long-Term, Multi-Lineage Engraftment InVivo.

Overexpression of 5TFs (e.g., LCOR, HOXA9, HOXA5, RUNX1 and ERG) usinglentiviral transduction of hemogenic endothelial (HE) cells issufficient to generate HSPCs with multi-lineage engraftment (Sugimura etal., 2017). To induce TF expression using non-integrating vectors, anepisomal system previously developed by Okita et al. was adapted forreprogramming fibroblasts into PSCs (Okita et al., 2013). Twopolycistronic episomal vectors, pCXLE-L95 (LCOR-P2A-HOXA9-T2A-HOXA5) andpCXLE-RE (RUNX1-P2A-ERG), were generated by Gateway cloning of the TFopen reading frames, extracted from pINDUCER-21-R UNX1-P2A-ERG andpINDUCER-21-LCOR-P2A-HOXA9-T2A-HOXA5 vectors, into the pCXLE-gw backbone(FIG. 10A). 2A peptides were used for polycistronic co-expression.

hPSC-derived HE cells were isolated from embryoid bodies (EBs) after 8days of differentiation by magnetic cell isolation of a CD34⁺ population(FIG. 6A) (Ditadi and Sturgeon, 2016; Sugimura et al., 2017).Subsequently, cells were cultured in a combination of cytokines andmorphogens that induce an endothelial-to-hematopoietic transition (EHT)(Ditadi et al., 2015), and after 3 days infected or transfected thecells with lentiviral or episomal vectors (FIG. 6A), respectively.Expression of the 5TFs in HE cells was verified by quantitative reversetranscription polymerase chain reaction (qRT-PCR) (FIG. 10B). The dayafter infection or transfection, cells were injected intrafemorally intoimmune-deficient NOD.Cg-Kit^(W-41J)Tyr⁺Prkdc^(scid)Il2rg^(tm1Wjl)/ThomJ(NBSGW) mice to evaluate their engraftment and repopulation capacity.Mice transplanted with cells infected with lentiviral vectors(lenti-5TF) were treated with doxycycline for 2 weeks to induce TFexpression. Human CD45⁺ cells were detected in bone marrow of micetransplanted with HE cells transfected with episomes (epi-5TF) at 10weeks post-injection, demonstrating human blood cell engraftment in vivo(FIG. 6B). In a second cohort of mice, human CD45⁺ cells were detectedin bone marrow of animals transplanted with epi-5TF cells at 16 weeks,thereby revealing the long-term engraftment potential of the cells (FIG.6B). Mice injected with human CD34⁺ umbilical cord blood cells (UCBs)and sacrificed at 10 and 16 weeks were used as reference for engraftment(FIG. 6B-6D).

Using a standard of human bone marrow chimerism ≥0.01%, it was foundthat 4 of 12 mice transplanted with lentivirally-derived cells wereengrafted at 10 weeks (FIGS. 6B and 6C). In a second cohort of mice,another 4 out of 12 mice were engrafted at 16 weeks (FIGS. 6B and 6C).In side-by-side experiments with separate cohorts of mice, engraftmentwas detected in 7 of 12 mice transplanted with epi-5TF cells at 10weeks, and 9 of 11 at 16 weeks (FIGS. 6B and 6C). Human CD45⁺ cells weredetected in the bone marrow of both the injected leg and thecontralateral femur of some of the mice engrafted with epi-5TF cellsanalyzed at 10 and 16 weeks post-transplant, indicating the capacity ofthese cells to migrate and repopulate distant niches in the bone marrow(FIGS. 6C and 6D).

Using human CD34⁺ cord blood HSPCs engrafted in NBSGW mice as areference, myeloid (CD33+), B (CD19⁺) and T (CD3⁺) cells were detectedin 3 out of 4 engrafted mice analyzed at 10 weeks, and 1 out of 4engrafted mice analyzed at 16 weeks post injection with lenti-5TF cells(FIG. 6D). Among the animals transplanted with epi-5TF cells, 6 out of 7and 5 out of 9 engrafted mice evaluated at 10 and 16 weeks,respectively, showed multi-lineage engraftment (FIG. 6D). Multi-lineagecapacity of HSPCs derived from hPSC-hemogenic endothelium transfectedwith epi-5TFs was further validated by an extendedfluorescence-activated cell sorting (FACS) analysis of bone marrow fromprimary engrafted NBSGW mice, which revealed human CD45⁺ cells, HSPCs(CD34+CD38−), neutrophils (PECAM+CD15+), T (CD3+/CD4, CD8), B(IgM+CD19⁺) and B progenitor cells (IgM-CD19⁺⁾ (FIGS. 6E and 11A-11E).These results indicated the multi-lineage differentiation and long-termengraftment potential of epi-5TF cells.

Episomal Vectors are Lost from hPSC-Derived-5TF Cells.

The episomal plasmids used herein carry the OriP and EBNA1 components ofthe Epstein-Barr virus that allow for self-replication of the vectors(Okita et al., 2013). GFP⁺ cells were sorted from HE cells transfectedwith epi-5TFs 48 hours after transfection to quantify the initial copynumber of plasmids per genome (FIG. 7A). To evaluate the presence ofepisomes within the engrafted cells, DNA was extracted from human CD45⁺cells sorted from bone marrow of primary transplanted mice sacrificed at6, 10 and 16 weeks after injection, and analyzed by droplet digitalpolymerase chain reaction (ddPCR) to detect cytoplasmic episomal DNA orthe presence of episomal DNA that might have integrated into the donorcells' genome (FIG. 7A). Engrafted cells from 4 of 5 mice analyzed at 6weeks had more than one copy of EBNA1 per genome, whereas none of thesamples sorted from the bone marrow of mice sacrificed at 10 (n=6) or 16(n=6) weeks revealed presence of the episomal vectors over the levels ofthe target reference gene (CD90) (FIGS. 7B and 7C). Additionally, as HEcells were transfected with pCXLE-EGFP together with the polycistronicvectors, GFP signal was evaluated within the human CD45⁺ populationfound in recipient bone marrow by FACS. Although less sensitive than theddPCR analysis, this independent approach identified GFP⁺ cells in 2 outof 5 mice examined at 6 weeks, but no positive signal in cells sortedfrom the recipient bone marrow at 10 (n=6) or 16 (n=6) weeks aftertransplantation (FIG. 12). Together, these results indicate loss of theepisomal vectors in vivo in engrafted cells that persist up to 16 weeksin engrafted primary animals.

Limiting-Dilution Analysis Reveals HSPCs Frequency and Self-RenewalCapacity of the Episomal-5TF-HSPCs Obtained from hPSCs.

To explore the self-renewal ability of the epi-5TF-HSPCs and quantifytheir frequency, human CD34⁺ cells were purified by magnetic-activatedcell sorting (MACS) from the bone marrow of multi-lineage engrafted miceand transplanted into secondary recipient NBSGW mice at a dose of 5,000,10,000 or 30,000 cells (FIG. 8A). Transplantation of 30,000 and 10,000human CD34⁺ cells from cord blood, lenti-5TF or epi-5TF primary injectedmice resulted in human CD45⁺ cells engrafted in bone marrow of secondaryrecipients analyzed at 10 weeks, while none of the animals injected with5,000 cells showed engraftment (FIG. 8B). More precisely, 5 of 8 micewhich had undergone secondary transplantation with 30,000 lenti-5TFcells, 9 of 13 injected with 30,000 epi-5TF, and 6 of 8 with 30,000 cordblood-derived cells revealed human CD45⁺ chimerism ≥0.01% in the bonemarrow (FIGS. 8B and 3C). Notably, chimerism of human CD45⁺ cells ≥0.01%was also identified in the contralateral leg of 4 out of 9 secondarymice engrafted with 30,000 human CD34⁺ cells derived from epi-5TFprimary transplanted mice (FIGS. 8C and 8D).

It was also documented herein that multi-lineage hematopoiesis waspresent in secondary recipient mice (FIG. 8D). An extended FACS analysisof bone marrow from secondary NBSGW mice engrafted with epi-5TF-derivedcells demonstrated the presence of human CD45⁺ cells, HSPCs(CD34+CD38−), neutrophils (PECAM+CD15), T (CD3+/CD4+, CD8+), B(IgM+CD19⁺) and B progenitor cells (IgM-CD19⁺) (FIGS. 8E and 11A-11E).These secondary transplantation results allowed for the determinationthat the HSPCs frequency of the epi-5TF cells was 1 in 40,659, which wassimilar to the 1 in 34,180 cells present in cord blood-derived cells,and almost 2-fold enhanced over the frequency of 1 in 75,479 present inrecipients of primary donor derived lentiviral-5TF cells (FIGS. 8F and14). Taken together, data presented herein indicate HSC-like potentialof the HE cells transfected with episomal-5TFs vectors.

Molecular Similarity of Mature Blood Cells Derived from Cord Blood andEpisomal-5TF Cells.

To evaluate the degree of similarity between the gene expressionprofiles of mature blood cell populations derived from epi-5TF cells andUCB, human CD34⁺ cells were isolated from the bone marrow of primarymulti-lineage engrafted mice by MACS. MACS-isolated cells were thencultured in methylcellulose supplemented with hematopoietic cytokines toform colony-forming units of mature blood cells in vitro (FIG. 9A).After 3 to 4 weeks in culture, human CD45⁺ cells representingdifferentiated myeloid cells were isolated by FACS and processed forin-droplet barcoding and single cell RNA-sequencing (FIG. 9A) (Klein etal., 2015). Unsupervised hierarchical clustering of epi-5TF and UCBsingle-cell transcriptomes revealed that epi-5TF and UCB cells clustertogether, indicating transcriptome-wide similarities (FIG. 9B).Graph-based clustering of single-cell transcriptomes was also performedand visualized subpopulation structure and cell-cell relationships usingt-Distributed Stochastic Neighbor Embedding (t-SNE) (FIGS. 9C and 9D)(Mateen and Hinton, 2008). This analysis allowed the identification ofnine transcriptionally distinct clusters for each sample (FIGS. 9C, 9D,13A and 13B). Gene ontology (GO) analysis of epi-5TF and cord bloodsubpopulation-specific gene signatures revealed enrichments forgranulocyte/neutrophil-related biological processes (FIGS. 9E and 9F),consistent with the myeloid lineage bias of standard CFU assays (Majetiet al., 2007). These analyses indicated the marked similarity of celltypes generated from both epi-5TF and cord blood samples.

Next, to quantitatively evaluate these molecular similarities andputative cell identities between cord blood and episomal-5TF-derivedcells, cluster-specific binary random forest (RF) classifiers werecreated based on cord blood clusters and signatures (FIG. 13B), whereeach RF learns how to distinguish one cluster relative to all others(Habib et al., 2017), and asked how similar epi-5TF cells are relativeto these cord blood clusters (FIG. 13C). This analysis indicated thatthe majority of epi-5TF-derived cells are highly similar to the mostabundant cord blood clusters (clusters 0 and 1), identified asgranulocytes/neutrophils based on GO analysis (FIGS. 9E, 9F, 13C and13D). There were also similarities, although less robust, betweenepi-5TF cells and cord blood clusters 2 and 4 (which are also putativegranulocyte cell types) (FIGS. 9F, 9G, 13C, 13D). Reconstruction of agranulocyte differentiation trajectory using CellRouter (Lummertz daRocha et al., 2018) revealed complex expression patterns, with not onlygenes upregulated along the trajectory enriched for granulocyte-relatedGO terms, but also genes downregulated or transiently up ordownregulated, capturing the complex nature of differentiation (FIG.13E-13G). Top genes dynamically regulated along this cell fatetransition revealed that cells along this trajectory express a gradientof marker genes, such as S100A8 and MMP9 (FIG. 13H-13J). Furthermore,binary RF models were used to assign a specific cord blood class to eachepi-5TF cell. This analysis demonstrated, for example, that ˜60% ofepi-5TF cells in cluster 1, 5 and 7 are classified as belonging to thecord blood cluster 0, ˜60% of cells in episomal clusters 0 and 6 and˜80% of cells in episomal clusters 3 and 4 are classified as cord bloodcluster 1 (FIG. 9G). Taken together, these analyses indicated that themost abundant myeloid cell types generated by either cord blood orepisomal-5TF cells following methylcellulose culture, show highlycomparable transcriptional profiles, indicating a high degree ofmolecular similarity.

Discussion

In work presented herein HSC-like cells were derived from human PSCsusing a combination of morphogen-directed differentiation and cell fateconversion by transient expression of 5 transcription factors. Buildingupon prior studies that establish definitive hemogenic endothelium asthe origin of HSCs (Bertrand et al., 2010; Boisset et al., 2010;Dieterlen-Lievre, 1975; Dzierzak and Speck, 2008; Ivanovs et al., 2017;Ivanovs et al., 2011), and the derivation of definitive HE from humanpluripotent stem cells (Ditadi et al., 2015; Kennedy et al., 2012), itwas verified herein that enforced expression of TFs LCOR, HOXA9, HOXA5,RUNX1 and ERG using polycistronic episomal vectors can generatetransgene-free HSPCs with long-term multi-lineage reconstitution andself-renewal potential.

Generation of HSC-like cells has been reported with combinations of TFsor even just a single factor (Lis et al., 2017; Sugimura et al., 2017;Tan et al., 2018; Tsukada et al., 2017). In prior work the transcriptionfactor cocktail of LCOR, HOXA9, HOXA5, RUNX1 and ERG (5TFs) wasdescribed as sufficient to generate HSPCs from hPSCs-derived HE cells(Sugimura et al., 2017). However, these cells were generated usingintegrating lentiviral vectors, and doxycycline addition was required toactivate the expression of the 5TFs. With the goal of creating improvedand safer HSPCs for translational approaches, the expression of thesesame 5TFs was induced in HE cells using polycystronic non-integratingepisomal vectors. Herein its is demonstrated that despite complete lossof episomal constructs through cell divisions between 6 to 10 weeksafter their transplantation, episomal-5TF-derived cells function as HSCsin vivo, maintaining multi-lineage differentiation and secondaryengraftment potential.

Human HSCs are defined by two major properties: multipotency, or theability to generate all blood cell lineages, and long-term self-renewal,characterized by the capacity of these cells to achieve serialtransplantations (Bhatia et al., 1997; Cashman et al., 1997; Hogan etal., 2002; Majeti et al., 2007). Accordingly, transplantation ofepisomal-5TF-HE-derived cells into secondary recipients has proven to besuccessful, with the identification of multi-lineage human CD45⁺ cellsin the bone marrow of injected mice at limiting dilution, indicatingclonal repopulation. Notably, engrafted cells derived from episomal-5TFcells can be found in the contralateral femur, indicating that themigration and homing potential of these cells is preserved, furtherreinforcing the presence of functional stem cell properties inepi-5TF-derived cells.

To evaluate the frequency of HSPCs from episomal-5TF, lentiviral-5TF orcord blood-derived cells, a limiting-dilution assay was performed, anapproach widely used for quantifying biological units such as stem cells(Huang et al., 2016; Szilvassy et al., 2002) ENREF_5. Interestingly,despite the fact that the percentage of human engrafted cells in bonemarrow of primary engrafted mice transplanted with cord blood is higherthan with episomal-5TF cells, the frequency of secondary repopulatingunits in the CD34+ population of engrafted cells is comparable,indicative of a similar stem cell potential between these cells.

Although previous work described the ability of hPSC-derived HSPCs toproduce mature blood cells in vivo, global transcriptomics analysisshowed that lentiviral-HE-derived cells clustered closest to HE cells,indicating that cells obtained with the lentiviral strategy may not becompletely converted to a HSC-like state, compromising their ability toproduce mature blood cells molecularly similar to UCB derivatives(Sugimura et al., 2017). Thus, single-cell RNA sequencing profiling wasperformed in mature cells derived from episomal-5TF and cord bloodcells. By using inDrop single-cell RNA-seq (Klein et al., 2015), a closetranscriptional resemblance was observed between mature blood cellsderived from episomal-5TF and cord blood cells after in vitromethylcellulose culture, which assesses directly for the first time theability of the system described herein to produce terminallydifferentiated mature effector blood cells whose gene expressionprofiles are comparable to cord blood derived cells.

Work described herein further supports the use of harnessing HSPCsderived from hPSCs for personalized cell therapies. Using these episomal5TF-hPSC-derived cells, modeling of human blood disorders and evaluatingand optimizing therapeutic strategies for HSC production is nowconceivable.

Materials and Methods

Cell Lines.

Experiments were performed with human CD34⁺ umbilical cord blood cellspurchased from AllCells, H9 human embryonic stem cells (ESC) (WiCell)and human iPSC lines (1157-iPSC, 1157-2-iPSC, and 1045-iPSC) generatedby the human Stem Cell Core Facility at Boston Children's Hospital (BCH)from peripheral blood mononuclear cells (PBMNCs) of healthy donorsreprogrammed using Epi5™ Episomal iPSC Reprogramming Kit (Thermo FisherScientific).

Mice.

NOD.Cg-Kit<W-41J>Tyr<+>Prkdc<scid>I12rg<tmlWjl>/ThomJ (NBSGW) mice (TheJackson Laboratory) were bred and housed at the BCH animal carefacility. All the animal experiments were performed in accordance toinstitutional guidelines approved by BCH animal care committee.

Episomal and Lentiviral Plasmids.

Polycistronic 5TFs-episomal plasmids were constructed from thepreviously described lentiviral pINDUCER-21-R UNX1-P2A-ERG andpINDUCER-21-LCOR-P2A-HOXA9-T2A-HOXA5 vectors (Addgene plasmids #97045and #97044) by Gateway-recombination with pCXLE-gw plasmid (Addgeneplasmid #37626) (Okita et al., 2013; Sugimura et al., 2017). Theresulting polycistronic vectors were named: pCXLE-L95, containing thefragment (LCOR-P2A-HOXA9-T2A-HOXA5); pCXLE-RE, containing the fragment(RUNX1-P2A-ERG). Episomal vectors (pCXLE-L95, pCXLE-RE, pCXLE-EGFP andpCXWB-EBNA1) were co-transfected into HE cells on day 3 of endothelialto hematopoietic transition (EHT) culture using Lipofectamine® LTX withPlus™ Reagent (Thermo Fisher Scientific) (1.5 μg of DNA and 2 μl ofLipofectamine per well) following manufacturer's instructions. pCXLE-gw,pCXLE-EGFP and pCXWB-EBNA1 were a gift from Shinya Yamanaka (Addgeneplasmids #37626, #27082 and #37624) (Okita et al., 2011; Okita et al.,2013).

HEK 293T/17 cells (ATCC) were transfected with the second-generationpackaging plasmids pMD2.G and psPAX2 (A gift from Didier Trono, Addgene#12259 and #12260) with either pINDUCER-21-RE or pINDUCER-21-L95 usingX-tremeGENE 9 (Sigma-Aldrich) to produce lentiviral particles. 36 and 60hours after transfection, virus were harvested, filtered with a 0.45 msyringe filter, and concentrated by ultracentrifugation at 23,000 r.p.m.for 2 hours at 4° C. Viruses were then reconstituted with 50 μl of EHTculture medium and titered by serial dilution on HEK 293T/17 cells usingGFP as indicator. Infection of HE cells was done in 200 μl of EHT mediasupplemented with Polybrene (8 μg ml⁻¹, Sigma) on day 3 of EHT cultureand the multiplicity of infection was 2.0 for each lentiviral plasmid.

hPSC Culture.

Human iPSCs and ESCs were maintained using human ESC-qualified Matrigel(BD) in mTeSR1 media (STEMCELL Technologies). Media were changed dailyand cells were passaged at 1:8 ratio every 7 days using standard clumppassaging techniques with Dispase (STEMCELL Technologies) or Versene(Thermo Fisher Scientific). Prior to initiation of EBs differentiation,colonies were expanded for 6 to 7 days over mouse embryonic fibroblasts(MTI-GlobalStem or Gibco, by Life Technologies) in DMEM/F12 (STEMCELLTechnologies) supplemented with 20% KnockOut Serum Replacement (KOSR)(Invitrogen), 1 mM L-glutamine (Life Technologies), 1 mM nonessentialamino acids (NEAA) (Life Technologies), 0.1 mM 3-mercaptoethanol (LifeTechnologies), and 10 ng/mL basic fibroblast growth factor (bFGF) (LifeTechnologies).

Embryoid Body Differentiation.

Embryoid body differentiation was performed as previously described(Ditadi and Sturgeon, 2016; Sugimura et al., 2017). Briefly, hPSCcolonies were incubated with 0.05% trypsin-EDTA for 5 min at 37° C. anddissociated to form small aggregates that were then washed andresuspended in StemPro-34 (Invitrogen, 10639-011) supplemented withL-glutamine (2 mM), human holo-Transferrin (150 μg mL⁻¹, Sigma T0665),monothioglycerol (MTG, 0.4 mM; Sigma), penicillin/streptomycin (10 ngmL⁻¹; Life Technologies), ascorbic acid (1 mM; Sigma) (indicated as“supplemented StemPro-34”), Y-27632 (10 μM; StemCell Technologies Inc.)and BMP4 (10 ng mL⁻¹). Cells were distributed into non-adherent 100 mmEZSPHERE dishes (EZSPHERE, Asahi Glass, ReproCELL; well size diameter400-500 μm, depth 100-200 μm, 14,000 wells per dish) at a density ofapproximately 5 million per dish. After 24 hours, bFGF (LifeTechnologies) and BMP4 were added to the media of each plate to make afinal concentration of 5 ng mL⁻¹ for each cytokine. On day 2, EBs wereharvested, pelleted, and resuspended in supplemented StemPro-34 withBMP4 (10 ng mL⁻¹), bFGF (5 ng mL⁻¹; Life Technologies), SB431542 (6 μM;STEMCELL-Technologies Inc.) and CHIR99021 (3 μM; STEMCELL-TechnologiesInc.). On day 3, medium was replaced with supplemented StemPro-34 withbFGF (5 ng mL⁻¹) and VEGF (15 ng mL⁻¹; R&D Systems). 72 hours later, EBswere harvested and resuspended in supplemented StemPro-34 with bFGF (2.5ng mL⁻¹), VEGF (7.5 ng mL⁻¹), SCF (100 ng mL⁻¹), EPO (2 U mL⁻¹; Amgen),IGF-1 (25 ng mL⁻¹), interleukin (IL)-11 (5 ng mL⁻¹) and IL-6 (10 ngmL⁻¹). Throughout the process of EB formation, cultures were kept in amulti-gas incubator set at 5% CO₂ 5% O₂ 90% N₂ and 37° C. Allrecombinant factors were human and purchased from Peprotech unlessspecifically indicated.

Isolation of HE and EHT Culture.

EBs were collected on day 8 of EB differentiation and washed withphosphate-buffered saline (PBS) before dissociation with 0.05% oftrypsin-EDTA (Thermo Fisher Scientific) for 5 min at 37° C. EBs werethen pipetted up and down with a 5 mL pipette to generate a single-cellsuspension and washed with PBS+2% of fetal bovine serum (FBS). Cellswere filtered through a 70 μm filter and incubated with CD34 MicrobeadKit (Miltenyi Biotec, 130-046-702) for 30 minutes on ice followingmanufacturer's indications. After incubation, CD34⁺ cells were isolatedusing magnetic cell isolation with LS columns (Miltenyi Biotec,130-042-401). Sorted cells were resuspended in supplemented StemPro-34media containing TPO (30 ng ml⁻¹), Y-27632 (10 μM; STEMCELL TechnologiesInc.), IL-6 (10 ng mL⁻¹), SCF (100 ng mL⁻¹), IL-3 (30 ng mL⁻¹), VEGF (5ng mL⁻¹), IGF-1 (25 ng mL⁻¹), IL-11 (5 ng mL⁻¹), bFGF (5 ng mL⁻¹),Flt-3L (10 ng mL⁻¹), BMP4 (10 ng mL⁻¹), EPO (2 U mL⁻¹), angiotensin II(10 μg L⁻¹; Sigma Aldrich), SHH (20 ng mL⁻¹) and angiotensin II receptortype I (AGTR1) blocker losartan potassium (100 μM; Fisher Scientific)and seeded into 24-well plates pre-coated with Growth Factor ReducedMatrigel (Fisher Scientific) at a density of 2.5×10⁴ to 5×10⁴ cells. Allrecombinant factors were human and purchased from Peprotech, unlessspecifically indicated.

Mice Transplantation and Analysis.

On day 4 of EHT culture, HE cells transfected or infected with episomalor lentiviral 5TFs were carefully washed with PBS and incubated withAccutase (STEMCELL Technologies) 3 minutes at 37° C. Cells werecollected and wells were washed twice with PBS+2% of FBS to recover allthe cells. 0.8×10⁵ to 2.0×10⁵ range of cells resuspended in PBS+2% ofFBS were intrafemorally transplanted into 6-10 weeks oldNOD.Cg-Kit<W-41J>Tyr<+>Prkdc<scid>Il2rg<tm1Wjl>/ThomJ (NBSGW) female ormale mice (The Jackson Laboratory) in a 10 μl volume using a 28.5-gaugeinsulin needle (McIntosh et al., 2015). Mice transplanted withapproximately 20,000 human CD34⁺ umbilical cord blood cells were used asa reference for engraftment. Prior to transplantation, mice were sedatedwith isoflurane and their femur was cannulated using a 26-half-gaugeneedle. Primary transplanted mice with lentiviral-5TF-derived cells werefed with Doxycycline Rodent Diet (Envigo-Teklad Diets; 625 p.p.m.) andDoxycycline (1.0 mg mL⁻¹) was added to the drinking water to maintaintransgene expression in vivo for 2 weeks. Sulfatrim was added to thedrinking water together with the doxycycline. Secondary transplantationfor limiting dilution assay was performed with increasing doses of5,000, 10,000 or 30,000 human CD34⁺ cells injected intrafemorally intounirradiated female NBSGW mice. Human CD34⁺ cells were isolated usingmagnetic cell isolation with CD34 microbeads from the bone marrow ofinjected and contralateral leg of primary engrafted mice showing ≥0.01%of multi-lineage human chimerism. Cells were kept on ice untilinjection. Mice were sacrificed at indicated time points, and femur andtibiae from the injected leg and contralateral leg were collected foranalysis. Single cell suspension was prepared using cell dissociationtechniques in PBS+2% FBS. Samples were lysed with Red Blood Cell LysingBuffer Hybri-Max™ (Sigma). From the cell suspension, 100-150 μl(approximately 5×10⁵-1×10⁶ cells) were stained in a total volume of 200μl of staining buffer (PBS+2% FBS).

qRT-PCR and ddPCR Analysis.

GFP⁺ cells were sorted 48 hours after infection or transfection of HEwith lentiviral or episomal 5TFs. Lentiviral-5TF-infected cells wereincubated 24 hours with doxycycline (2 μg mL⁻¹). Total RNA was isolatedfrom GFP⁺ cells and HE cells without infection or transfection, usingPicopure RNA isolation kit, following the manufacturer's instructions(Thermo Fisher Scientific). cDNA was synthesized with SuperScript VILOcDNA Synthesis Kit (Invitrogen). Quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) was carried out in duplicate ortriplicate for each sample using Power SYBR green PCR Master Mix(Applied Biosystems). Gene expression was normalized using GAPDH asendogenous control. The following oligonucleotides were used in thestudy; HOXA9 fwd: 5′-TGTACCACCACCATCACCAC-3′ (SEQ ID NO: **), HOXA9_rev:5′-CAGCGGTTCA GGTTTAATGC-3′ (SEQ ID NO: **) (Integrated DNATechnologies); HOXA5_fwd: 5′-GGCTACAATGG CATGGATCT-3′ (SEQ ID NO: **);HOXA5_rev: 5′-GCTGGAGTTGCTTAGGGAGTT-3′ (SEQ ID NO: **) (Integrated DNATechnologies); LCOR_fwd: 5′-CACTTCCCTGAGCCACTCTC-3 (SEQ ID NO: **);LCOR_rev: 5′-TGGAGTGTCCAAAACCTTCC-3′ (SEQ ID NO: **) (Integrated DNATechnologies); RUNX1 (QT00026712, Quiagen QuantiTect); ERG1 (QT00074193,Quiagen QuantiTect) and GAPDH (QT00079247, Quiagen QuantiTect).

For droplet digital polymerase chain reaction (ddPCR) analysis, humanCD45+ cells were sorted from murine bone marrow at the indicated timepoints or GFP+ cells were sorted within HE cells 48 hours after thecell's transfection. Cells were pelleted and resuspended in a solutionof 25% DirectPCR Lysis Reagent (Viagen #301-C) and 75% v/v water withProteinase K Solution (Viagen #501-PK) to a final concentration of 400μg/mL. Lysis was carried out at 56′C for two hours, then the reactionwas inactivated at 85° C. for 45 minutes. ddPCR reactions were preparedusing ddPCR SuperMix for Probes (No dUTP) (Biorad #186-3023). 25 μLreactions were prepared comprised of 12.5 μL SuperMix, 1.25 L FAMprobeset, 1.25 μL HEX probeset, 2.5 μL DNA (viagen lysate) and 7.5 μLwater. For analysis of sorted GFP'⁰ cells, the viagen lysate was diluted1:800-1:1000. Reactions were mixed thoroughly and droplets weregenerated using the QX100 droplet generator (Biorad) per manufacturer'sinstruction. 20 L of droplet suspension was used for PCR and analysis.PCR settings were as follows: 95C-10 minutes, 94′C-30 seconds, 58.1°C.-1 minute, 98′C-10 minutes, with steps 2 and 3 repeated 40 times.After PCR, samples were analyzed using the QX100 droplet reader (Biorad)and the CNV2 assay setting. Assay sensitivity and technical run qualitywas confirmed using pCXLE-RE plasmid digested with EcoRI (NEB #R0101)and spiked into a 20 ng/μL solution of human genomic DNA in water(Promega #G1471) at known concentrations. Standard samples containing 0,6 and 6000 plasmids per reaction volume were run in parallel with eachexperimental sample. Thresholds for positive signal were determinedbased on standard samples and were the same for all samples in each run.Copy number present per 20 μL reaction volume was calculated by thebuilt-in software (Biorad) and R/Bioconductor package was used forrepresentations as previously described (Chiu et al., 2017). Episomalplasmids were detected with FAM-labeled probeset targeting EBNA1which isunique to the plasmids. The probesets were as follow: EBNA1 F:GCTCACCATCTGGGCCAC (SEQ ID NO: **) P:/56-FAM/CCTCCAGGT/ZEN/AGAAGGCCATTTTTCCACCCTGTAG/3IABkFQ/R:TCATCATCATCCGGGTCTCC (SEQ ID NO: **) (Vo et al., 2016). HEX-labeledreference probes were designed to the human CD90 (Thy1) locus. hCD90 F:CAGAGGCTTGGTTTTATTGTGC (SEQ ID NO: **), P:/5HEX/CGGTGGTTC/ZEN/TTCCTGTTCTGTGACT/3IABkFQ/(SEQ ID NO: **), R:GGACACTT CTCAGGAAATGGCTTTT (SEQ ID NO: **). All probesets were orderedas IDT Standard PrimeTime qPCR assays (Integrated DNA Technologies).

Flow Cytometry.

Cells grown in culture or harvested from animal tissues were stainedwith 4:200-1:200 dilution of each antibody for at least 30 min on ice inthe dark, with the following antibody panels: Lineage panel, CD45 PE-Cy5(Beckman Coulter), CD33 APC (BioLegend), CD19 PE (BioLegend), CD3 PE-Cy7(BioLegend), mouse CD45.1-APC-Cy7 (BioLegend) and4′,6-diamidino-2-phenylindole (DAPI) (Fisher Scientific); HSPC panel,CD34 PE-Cy7 (BioLegend), CD38 PE-Cy5 (BioLegend), mouse CD45.1-APC-Cy7and DAPI; B cell panel CD45 PE-Cy5, CD19 PE, IgM BV510 (BioLegend), CD20PE-Cy7 (BioLegend), mouse CD45.1-APC-Cy7 and DAPI; T cell panel, CD45 PE(BD Biosciences), CD3 PE-Cy7, CD4 PE-Cy5 (BioLegend), CD8 BV421 (BDBioscience), mouse CD45.1-APC-Cy7 and DRAQ7 (BioLegend); neutrophilpanel CD45 PE-Cy5, CD15 APC (BioLegend), PECAM (CD31) PE (BDBioscience), mouse CD45.1-APC-Cy7 and DAPI.

Cord blood mononuclear cells (MNCs) (AllCells) stained with individualantibodies were used as a positive control for antibody staining andcompensation. To determine the gating, bone marrow from human cord bloodengrafted mice was used as a control and fluorescence minus one (FMO)controls were performed with bone marrow of episomal-5TFs engraftedmice. Unless specifically indicated, all the antibodies used are againsthuman cells. Acquisitions were done on BD FACSAria II cell sorter or BDLSRFortessa cytometer. Sorting was performed on a BD FACSAria II cellsorter. Flow cytometry data were analyzed using FlowJo V.10.

Single-Cell RNA-Seq Using inDrop Technology.

Engrafted human CD34+ cells were isolated from the bone marrow ofepisomal-5TF (n=2) or cord blood (n=2) injected NBSGW mice usingmagnetic cell isolation as previously introduced. 2×10³ to 54×10³ cellswere resuspended into 3 mL of methylcellulose (H4434; StemCellTechnologies) supplemented with IL-6 (10 ng mL⁻¹), Flt-3L (10 ng mL⁻¹),TPO (50 ng mL⁻¹), EPO (2 U mL⁻¹) and 30 μl of a 100× concentrate inpenicillin/streptomycin. The cell's suspension was then plated into 60mm plates and kept in a humidified chamber at 37° C. and 5% of CO₂.After 3 to 4 weeks, from each sample, several individual colony-formingunits of granulocyte-macrophage (CFU-GM) were picked manually, combined,and washed with PBS+2% FBS to remove the methylcellulose. HumanCD45+mouse CD45-negative and DAPI-negative live cells were thenFACS-sorted and processed for inDrop barcoding at the single-cell coreof the Harvard Medical School.

Thus, approximately 6,000 cells from each condition were encapsulatedand libraries were prepared the same day, with the same stock ofprimer-gels and RT-mix. Transcriptome barcoding and librariespreparation for single-cell messenger ribonucleic acid (mRNA) sequencingwas performed using the most up-to-date inDrop protocol (Zilionis etal., 2017). Libraries were sequenced on an Illumina NextSeq 500sequencer using a NextSeq High 75 cycle kit: 61 cycles for read 1, 8cycles for index i7 read, 8 cycles for index i5 read, and 14 cycles forread 2. Raw sequencing reads were processed using the inDrop pipeline(which can be found on the world wide web atwww.github.com/indrops/indrops) using default parameters.

ELDA software analysis.

To estimate the frequency of HSPCs from episomal-5TF, lentiviral-5TF andcord blood cells, the number of mice showing multi-lineage engraftmentwith human CD45⁺ cells ≥0.01% was evaluated for injection of 5,000,10,000 and 30,000 cells, and the log fraction of non-engrafted(non-responding) mice was plotted relative to cell dose. Number of miceinjected and engrafted with each condition is indicated in Resultssection, legend of FIGS. 8A-8F and 14. Data obtained from the limitingdilution assay were analyzed using ELDA software (Hu and Smyth, 2009).

Single-Cell inDrop RNA-Seq Quality Control and Downstream Analysis.

To analyze Single-cell inDrop RNA-seq data quality control,dimensionality reduction, clustering and differential expression wasperformed analysis using the R package Seurat (Satija et al., 2015).Next, cord blood and epi-5TF samples were separately analyzed andapplied the same quality control metrics. All genes that were notdetected in at least 30 cells were excluded. All cells with less than500 genes detected were excluded. As expression of ribosomal ormitochondrial genes was shown to be markers of technical variation insingle-cell RNA-data (Ilicic et al., 2016), cells were removed where theproportion of the transcript counts derived from mitochondrial genes wasgreater than 10%. Among the cells remaining after quality control, inthe combined dataset, the median number of genes detected per cellcontrol was 5,028. 6,375 cells were analyzed (out of 9,492 cells intotal) and 32,041 genes fulfilled quality control metrics. For theanalysis of individual datasets, 3,055 cord blood-derived cells (total:4,587 cells) were analyzed and 31,567 genes (total: 41,569 genes) wherethe median number of detected genes per cell was 5,736. In epi-5TF cells(total: 4,905 cells), 3,321 cells remained after quality control and themedian number of genes detected per cell was 4,231 with 32,221 genes(out of 41,569) passing the quality control metrics.

Transcript counts were normalized by using a global scalingnormalization method “LogNormalize” that normalizes gene expressionmeasurements for each cell by the total expression, multiplies this by ascale factor of 10,000, and log-transforms the result, as implemented inthe package Seurat. Next, a set of most variable genes were identifiedacross all cells from the cord blood (1,313 genes) or the episomal-5TF(1,380 genes) conditions by calculating the average expression anddispersion for each gene, placing these genes into bins and calculatinga z-score for dispersion within each bin. Cell cycle scores werecalculated and predicted the cell cycle phase of each single cell inboth experimental conditions. Then, to reduce the effect of libraryquality and complexity, cell cycle effects and mitochondrial genecontent, this unwanted sources were regressed out of variation by usinga linear model. The z-score scaled residuals of this linear model wereused for dimensionality reduction and clustering. Next, a principalcomponent (PC) analysis were performed using the most variable genes andselected statistically significant components using a permutation test.For the cord blood-derived cells dataset, the first 31 PCs were usedwhile 21 PCs were used for the episomal-5TF-derived cells dataset.

These PCs were used for graph-based clustering to identify thesubpopulation structure of cord blood-derived and episomal-5TFsingle-cell transcriptomes. 9 subpopulations were identified in the cordblood and episomal datasets. The t-stochastic neighbor embedding (t-SNE)were used to visualize cell-cell relationships and the underlyingsubpopulation structure in a space of reduced dimensionality. Lastly,subpopulation-specific gene signatures were identified in each dataset.To annotate subpopulations identified from cord blood and episomal-5TFderived cells, gene ontology (GO) analysis was preformed using the genesignatures previously identified. The R package ClusterProfiler was usedfor GO analysis.

InDrop-Seq Clusters to Train RF Classifiers.

Cluster labels was identified by graph-based clustering to train randomforest (RF) classifiers on the cord blood dataset presented herein.These RF models were used to quantitatively compare the similarity ofepisomal-5TF and cord blood-derived cells. A RF classifier is a machinelearning approach based on an ensemble of decision trees, each trainedon a random “bag” of features (e.g. genes).

Cord blood-derived cells were used to train RF classifiers and evaluatethe congruence between cord blood and episomal-5TF cells subpopulations.Only clusters containing more than 90 cells were used to create atraining dataset. The training set was composed of 3,001 cells (˜98% ofthe cord blood dataset presented herein) and Seurat's scaled expressionvectors was used to train the RF classifiers. These models were used toclassify 3,321 episomal-5TF-derived cells and determine the similaritybetween the cell types generated by either cord blood or epi-5TF cells.Binary RF classifiers were then trained using 2,000 trees on the cordblood dataset using the R package randomForest. Cluster-specific genesignatures were identified by Seurat as features. Two independentstrategies were used for binary classification. In the first one, theprobability of indistinguishability of epi-5TF cells and each cord bloodsubpopulation relative to all other subpopulations such that eachepisomal cell has a probability to be indistinguishable from cord bloodsubpopulations (Figures S4C and S4D). In the second one, votes receivedby each episomal-5TFs cell were combined and assigned a label (one of 7possible labels) to each episomal-5TFs cell. A valid assignment wasdetermined only if over 15% of the trees in the forest contributed tothe majority vote to a particular cord blood cluster (FIG. 4G). Whenthis criterion is not fulfilled, episomal-5TFs cells were not assignedto any cord blood subpopulation.

Reconstitution of Granulocyte Single-Cell Trajectory Using CellRouter.

A granulocyte differentiation trajectory was reconstructed from epi-5TFcells selecting subpopulations on the “extreme ends” of the t-SNEanalysis (Subpopulation 8 and 5). It was noticed that subpopulation 5expresses high levels of the myeloid marker S100A8, and chose thesesubpopulation as the starting point of the granulocyte differentiationtrajectory. Genes were identified dynamically regulated along thistrajectory and clustered kinetic trends into 5 transcriptional clusters,which include genes monotonically up or downregulated as well as genestransiently up or downregulated. Gene ontology analysis using genes ineach transcriptional cluster was performed using the R packageclusterProfiler (Yu et al., 2012).

Data and Software Availability.

The single-cell RNA-seq data generated in this study are: inDrop singlecell RNA sequencing of mature cells differentiated in CFU conditionsfrom integration-free hematopoietic cells derived from human pluripotentstem cells. The degree of similarity was evaluated between the geneexpression profiles of mature blood cells populations derived fromepi-5TF cells and UCB cells. The accession number for the RNA-seq datareported in this paper is GSE114339.

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1. A method for making hematopoietic stem cells (HSCs) and hematopoieticstem and progenitor cells (HSPCs) comprising in vitro transfectinghemogenic endothelia cells (HE) with an exogenous gene coding copy of atleast one of the following transcription factors ERG, HOXA9, HOXA5, LCORand RUNX1 comprised in a non-integrative vector, wherein thetranscription factors are expressed in the transfected cells to producea population of multilineage HSCs and HSPCs that engrafts in recipienthost after implantation.
 2. A method of making hematopoietic stem cells(HSCs) and hematopoietic stem and progenitor cells (HSPCs) comprising:a) generating embryonic bodies (EB) from pluripotent stem cells; b)isolating hemogenic endothelia cells (HE) from the resultant populationof EB; c) inducing endothelial-to-hematopoietic transition (EHT) inculture in the isolated HE to obtain hematopoietic stem cells, and d) invitro transfecting the induced HE with an exogenous gene coding copy ofat least one of the following transcription factors ERG, HOXA9, HOXA5,LCOR and RUNX1 comprised in a non-integrative vector.
 3. The method ofclaim 1 or 2, wherein the method is an in vitro method.
 4. The method ofany one of claims 1-3, wherein the EB are generated or induced frompluripotent stem cells (PSC) by culturing or exposing the PSC tomophogens for about 8 days.
 5. The method of claim 4, wherein themophogens selected from the group consisting of Holo-Transferrin,mono-thioglycerol (MTG), ascorbic acid, bone morphogenetic protein(BMP)-4, basic fibroblast growth factor (bFGF), SB431542, CHIR99021,vascular endothelial growth factor (VEGF), interleukin (IL)-6,insulin-like growth factor (IGF)-1, interleukin (IL)-11, stem cellfactor (SCF), erythropoietin (EPO), thrombopoietin (TPO), interleukin(IL)-3, and Fms related tyrosine kinease 3 ligand (Flt-3L).
 6. Themethod of any one of claims 1-5, wherein the EBs are less than 800microns in size and are selected.
 7. The method of any one of claims1-6, wherein the EB cells within the EBs are compactly adhered to eachother and requires trypsin digestion in order to dissociate the cells toindividual cells.
 8. The method of claim 6 or 7, wherein the EB cells ofthe selected EBs are dissociated prior to the isolation of HE.
 9. Themethod of any one of claims 1-8, wherein the population of PSC isinduced pluripotent stem cells (iPS cells) or embryonic stem cells(ESC).
 10. The method of claim 9, wherein the induced pluripotent stemcells are produced by introducing only reprogramming factors OCT4, SOX2,KLF4 and optionally c-MYC or nanog and LIN28 into mature cells.
 11. Themethod of claim 10, wherein the mature cells are selected from the groupconsisting of B lymphocytes (B-cells), T lymphocytes, (T-cells),fibroblasts, and keratinocytes.
 12. The method of claim 8, 9 or 10,wherein the induced pluripotent stem cells are produced by introducingthe reprogramming factors two or more times into the mature cells. 13.The method of any one of claims 1-12, wherein the HE are definitive HE.14. The method of any one of claims 1-13, wherein the HE are isolatedimmediately from selected and dissociated EB.
 15. The method of any oneof claims 1-14, wherein the HE are FLK1+, CD34+, CD43−, and CD235A−.(these biomarkers are those on HE before theendothelial-to-hematopoietic transition?)
 16. The method of any one ofclaims 1-15, wherein the hematopoietic cells are CD34+ and CD45+. 17.The method of any one of claims 1-16, wherein theendothelial-to-hematopoietic transition occurs by culturing the isolatedHE in thrombopoietin (TPO), interleukin (IL)-3, stem cell factor (SCF),IL-6, IL-11, insulin-like growth factor (IGF)-1, erythropoietin (EPO),vascular endothelial growth factor (VEGF), basic fibroblast growthfactor (bFGF), bone morphogenetic protein (BMP)4, Fms related tyrosinekinase 3 ligand (Flt-3L), sonic hedgehog (SHH), angiotensin II, chemicalAGTR1 (angiotensin II receptor type I) blocker losartan potassium. 18.The method of any one of claims 1-17, wherein the multilineage HSCs areCD34+CD38−CD45+.
 19. The method of any one of claims 1-18, wherein themultilineage HSPCs are CD34+CD45+.
 20. The method of claim 1 or 2,wherein the non-integrative vector is an episomal vector.
 21. The methodof claim 1 or 2, wherein at least 2, at least 3, at least 4, or at least5 transcription factors are transfected.
 22. An engineered cell derivedfrom a population of HE and produced by a method of any one of claims1-21.
 23. The engineered cell of claim 22, wherein the engineered cellcomprises an exogenous copy of each of the following transcriptionfactors ERG, HOXA9, HOXA5, LCOR and RUNX1.
 24. The engineered cell ofclaim 22 or 23, wherein the engineered cell further comprises anexogenous copy of each of the following reprogramming factors OCT4,SOX2, KLF4 and optionally c-MYC.
 25. A composition comprising apopulation of engineered cells of any one of claims 22-24.
 26. Thecomposition of claim 25, further comprising a pharmaceuticallyacceptable carrier.
 27. A pharmaceutical composition comprising apopulation of engineered cells of any one of claims 22-24 and apharmaceutically acceptable carrier.
 28. A pharmaceutical composition ofclaim 27 for use in cellular replacement therapy in a subject.
 29. Amethod of cellular replacement therapy in a subject in need thereof, themethod comprising administering a population of engineered cells ofclaims 22-24, or a composition of claim 25-26, or a pharmaceuticalcomposition of claim 27 to a recipient subject.
 30. The method ofcellular replacement therapy of claim 29, wherein the subject is apatient who has undergone chemotherapy or irradiation or both, andmanifest deficiencies in immune function or lymphocyte reconstitution orboth deficiencies in immune function and lymphocyte reconstitution. 31.The method of cellular replacement therapy of claim 29 or 30, whereinthe subject prior to implantation, the immune cells are treated ex vivowith prostaglandin E2 and/or antioxidant N-acetyl-L-cysteine (NAC) topromote subsequent engraftment in a recipient subject.
 32. The method ofcellular replacement therapy of claim 29 or 30 or 31, wherein the immunecells are autologous to the recipient subject or at least HLA typematched with the recipient subject.